Methods for producing isobutene from 3-methylcrotonic acid

ABSTRACT

Described are methods for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene wherein said 3-methylcrotonic acid is obtained by the enzymatic conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid or wherein said 3-methylcrotonic acid is obtained by the enzymatic conversion of 3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid. It is described that the enzymatic conversion of 3-methylcrotonic acid into isobutene can, e.g., be achieved by making use of a 3-methylcrotonic acid decarboxylase, preferably an FMN-dependent decarboxylase associated with an FMN prenyl transferase, an aconitate decarboxylase (EC 4.1.1.6), a methylcrotonyl-CoA carboxylase (EC 6.4.1.4), or a geranoyl-CoA carboxylase (EC 6.4.1.5).

The present invention relates to methods for the production of isobutenecomprising the enzymatic conversion of 3-methylcrotonic acid intoisobutene wherein said 3-methylcrotonic acid is obtained by theenzymatic conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acidor wherein said 3-methylcrotonic acid is obtained by the enzymaticconversion of 3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid. Theenzymatic conversion of 3-methylcrotonic acid into isobutene can, e.g.,be achieved by making use of a 3-methylcrotonic acid decarboxylase,preferably an FMN-dependent decarboxylase associated with an FMN prenyltransferase, an aconitate decarboxylase (EC 4.1.1.6), amethylcrotonyl-CoA carboxylase (EC 6.4.1.4), or a geranoyl-CoAcarboxylase (EC 6.4.1.5). Further, said 3-methylcrotonyl-CoA can beobtained by the enzymatic conversion of 3-methylglutaconyl-CoA into3-methylcrotonyl-CoA.

A large number of chemical compounds are currently derived frompetrochemicals. Alkenes (such as ethylene, propylene, the differentbutenes, or else the pentenes, for example) are used in the plasticsindustry, for example for producing polypropylene or polyethylene, andin other areas of the chemical industry and that of fuels.

Butylene exists in four forms, one of which, isobutene (also referred toas isobutylene), enters into the composition of methyl-tert-butyl-ether(MTBE), an anti-knock additive for automobile fuel. Isobutene can alsobe used to produce isooctene, which in turn can be reduced to isooctane(2,2,4-trimethylpentane); the very high octane rating of isooctane makesit the best fuel for so-called “gasoline” engines. Alkenes such asisobutene are currently produced by catalytic cracking of petroleumproducts (or by a derivative of the Fischer-Tropsch process in the caseof hexene, from coal or gas). The production costs are therefore tightlylinked to the price of oil. Moreover, catalytic cracking is sometimesassociated with considerable technical difficulties which increaseprocess complexity and production costs.

The production by a biological pathway of alkenes such as isobutene iscalled for in the context of a sustainable industrial operation inharmony with geochemical cycles. The first generation of biofuelsconsisted in the fermentative production of ethanol, as fermentation anddistillation processes already existed in the food processing industry.The production of second generation biofuels is in an exploratory phase,encompassing in particular the production of long chain alcohols(butanol and pentanol), terpenes, linear alkanes and fatty acids. Tworecent reviews provide a general overview of research in this field:Ladygina et al. (Process Biochemistry 41 (2006), 1001) and Wackett(Current Opinions in Chemical Biology 21 (2008), 187).

The conversion of isovalerate to isobutene by the yeast Rhodotorulaminuta has been described (Fujii et al. (Appl. Environ. Microbiol. 54(1988), 583)), but the efficiency of this reaction, less than 1millionth per minute, or about 1 for 1000 per day, is far frompermitting an industrial application. The reaction mechanism waselucidated by Fukuda et al. (BBRC 201 (1994), 516) and involves acytochrome P450 enzyme which decarboxylates isovalerate by reduction ofan oxoferryl group Fe^(V)=O. Large-scale biosynthesis of isobutene bythis pathway seems highly unfavourable, since it would require thesynthesis and degradation of one molecule of leucine to form onemolecule of isobutene. Also, the enzyme catalyzing the reaction usesheme as cofactor, poorly lending itself to recombinant expression inbacteria and to improvement of enzyme parameters. For all these reasons,it appears very unlikely that this pathway can serve as a basis forindustrial exploitation. Other microorganisms have been described asbeing marginally capable of naturally producing isobutene fromisovalerate; the yields obtained are even lower than those obtained withRhodotorula minuta (Fukuda et al. (Agric. Biol. Chem. 48 (1984), 1679)).

Gogerty et al. (Appl. Environm. Microbiol. 76 (2010), 8004-8010) and vanLeeuwen et al. (Appl. Microbiol. Biotechnol. 93 (2012), 1377-1387)describe the production of isobutene from acetoacetyl-CoA by enzymaticconversions wherein the last step of the proposed pathway is theconversion of 3-hydroxy-3-methylbutyric acid (also referred to as3-hydroxyisovalerate (HIV)) by making use of a mevalonate diphosphatedecarboxylase. This reaction for the production of isobutene from3-hydroxy-3-methylbutyric acid is also described in WO2010/001078. InGogerty et al. (loc. cit.) and in van Leeuwen et al. (loc. cit.) theproduction of 3-hydroxy-3-methylbutyric acid is proposed to be achievedby the conversion of 3-methylcrotonyl-CoA via3-hydroxy-3-methylbutyryl-CoA. In order to further improve theefficiency and variability of methods for producing isobutene fromrenewable resources, there is a need for alternative routes for theprovision of isobutene and its precursors.

The present invention meets this demand by providing a method for theproduction of isobutene comprising the enzymatic conversion of3-methylcrotonic acid (also termed 3-methyl-2-butenoic acid) intoisubutene.

The enzymatic conversion of 3-methylcrotonic acid into isobutene is adecarboxylation reaction. A decarboxylation is a chemical reaction thatremoves a carboxyl group and releases carbon dioxide (CO₂).

The decarboxylation of 3-methylcrotonic acid has already been suggestedin US-A1-2009/0092975 while there is no experimental evidence for thisconversion. In US-A1-2009/0092975, a nucleic acid sequence called PAD1derived from Saccharomyces cerevisiae is described and is disclosed toencode a decarboxylation enzyme. This enzyme is suggested to be usefulas a selectable marker in a recombinant organism while it is describedthat a “weak acid” may be used as the selecting agent. 3-methylcrotonicacid is mentioned, among many others, as a potential “weak acid”.

However, it was only later found that the above PAD1, in reality, doesnot provide for the decarboxylase activity.

In fact, the bacterial ubiD and ubiX or the homologous eukaryotic fdc1and pad1 genes have been implicated in the non-oxidative reversibledecarboxylation. The combined action of phenylacrylic acid decarboxylase(PAD) and ferulic acid decarboxylase (FDC) is considered to be essentialfor the decarboxylation of phenylacrylic acid in Saccharomycescerevisiae (J. Biosci. Bioeng. 109, (2010), 564-569; AMB Express, 5:12(2015) 1-5; ACS Chem. Biol. 10 (2015), 1137-1144).

Recently, the above enzyme family described as phenylacrylic aciddecarboxylase (PAD) was characterized as an FMN prenyl-transferase andno longer as a decarboxylase. It has been shown that Fdc1 (but not PAD)is solely responsible for the reversible decarboxylase activity and thatit requires a new type of cofactor, namely a prenylated flavinsynthesized by the associated UbiX (or Pad1) protein. Thus, the realenzymatic activity of this PAD enzyme has been identified as thetransformation of a flavin mononucleotide (FMN) cofactor with a prenylmoiety (from di-methyl-allyl-phosphate or pyrophosphate called DMAP orDMAPP).

Accordingly, in contrast to the prior art's belief, the realdecarboxylase is the ferulic acid decarboxylase (FDC) in associationwith the modified FMN (prenylated-FMN). This mechanism of the ferulicacid decarboxylase (FDC) in association with the modified FMN(prenylated-FMN) (the latter provided by the PAD enzyme) was recentlydescribed and involves a surprising enzymatic mechanism, i.e., anα,β-unsaturated acid decarboxylation via a 1,3-dipolar cyclo-addition.Moreover, the structure of this FDC decarboxylase has recently beenelucidated (Nature 522 (2015), 497-501; Nature, 522 (2015), 502-505;Appl. Environ. Microbiol. 81 (2015), 4216-4223).

The use of the above family of enzymes has previously been described forthe conversion of α-β unsaturated carboxylic acid into terminal alkenesin US-A1-2009/0092975 as mentioned above while WO2012/018624 is directedto microorganisms and methods for the biosynthesis of aromatics,2,4-pentadienoate and 1,3-butadiene and WO2013/028519 is directed tomicroorganisms and methods for producing 2,4-pentadienoate, butadiene,propylene, 1,3-butanediol and related alcohols.

Moreover, WO2013/186215 describes a method for preparing amono-unsaturated alkene comprising contacting an aliphaticmono-unsaturated carboxylic acid with an Fdc1 polypeptide and a Pad1polypeptide. However, in WO2013/186215, both, the Fdc1 polypeptide andthe Pad1 polypeptide are classified as enzymes having a decarboxylaseactivity.

In contrast, in the present invention, the above enzymes areartificially implemented in a pathway which ultimately leads to theproduction of isobutene. Thus, in a main aspect, the present inventionrelates to a method for the production of isobutene comprising theenzymatic conversion of 3-methylcrotonic acid into isobutene (step I asshown in FIG. 1),

wherein said method further comprises

-   -   (a) providing the 3-methylcrotonic acid by the enzymatic        conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid        (steps VIa, VIb or VIc as shown in FIG. 1), or    -   (b) providing the 3-methylcrotonic acid by the enzymatic        conversion of 3-hydroxyisovalerate (HIV) into 3-methylcrotonic        acid (step II as shown in FIG. 1)

Preferably, the enzymatic conversion of 3-methylcrotonic acid intoisobutene is achieved by making use of a 3-methylcrotonic aciddecarboxylase.

The method for the production of isobutene from 3-methylcrotonyl-CoA via3-methylcrotonic acid or from 3-hydroxyisovalerate (HIV) via3-methylcrotonic acid may be embedded in a pathway for the production ofisobutene starting from acetyl-CoA which is a central component and animportant key molecule in metabolism used in many biochemical reactions.The corresponding reactions are schematically shown in FIG. 1.

Therefore, the present invention also relates to pathways starting fromacetyl-CoA and leading to 3-methylcrotonic acid (which is thenultimately converted into isobutene) via two alternative pathways whichare schematically shown in FIG. 1 and will be explained in more detailfurther below.

The Routes for the Enzymatic Conversion from Acetyl-CoA into Isobutenevia Acetoacetyl-CoA and 3-Methylcrotonic Acid

The Enzymatic Conversion of 3-Methylcrotonic Acid into Isobutene: Step Ias Shown in FIG. 1

The enzymatic conversion of 3-methylcrotonic acid into isobutene isschematically shown in FIG. 2B.

According to the present invention, the enzymatic conversion of3-methylcrotonic acid (also termed 3-methyl-2-butenoic acid or3,3-dimethyl-acrylic acid) into isobutene (also termed isobutylene or2-methyl-propene) can be achieved by a decarboxylation.“Decarboxylation” is generally a chemical reaction that removes acarboxyl group and releases carbon dioxide (CO₂).

The enzymatic conversion of 3-methylcrotonic acid into isobutene canpreferably be achieved by making use of a 3-methylcrotonic aciddecarboxylase. In accordance with the present invention, a3-methylcrotonic acid decarboxylase is an enzyme which is capable ofconverting 3-methylcrotonic acid into isobutene in a decarboxylationreaction.

In preferred embodiments, the 3-methylcrotonic acid decarboxylase isselected from the group consisting of:

-   (i) an FMN-dependent decarboxylase associated with an FMN prenyl    transferase; or-   (ii) an aconitate decarboxylase (EC 4.1.1.6); or-   (iii) a methylcrotonyl-CoA carboxylase (EC 6.4.1.4); or-   (iv) a geranoyl-CoA carboxylase (EC 6.4.1.5).

Thus, according to one aspect, the enzymatic conversion of3-methylcrotonic acid into isobutene can preferably be achieved bymaking use of a 3-methylcrotonic acid decarboxylase, wherein said3-methylcrotonic acid decarboxylase is an FMN-dependent decarboxylaseassociated with an FMN prenyl transferase.

The enzymatic conversion of 3-methylcrotonic acid into isobuteneutilizing an FMN-dependent decarboxylase associated with an FMN prenyltransferase relies on a reaction of two consecutive steps catalyzed bythe two enzymes, i.e., the FMN-dependent decarboxylase (catalyzing theactual decarboxylation of 3-methylcrotonic acid into isobutene) with anassociated FMN prenyl transferase which provides the modified flavincofactor. The flavin cofactor may preferably be FMN or FAD. FMN (flavinmononucleotide; also termed riboflavin-5′-phosphate) is a biomoleculeproduced from riboflavin (vitamin B2) by the enzyme riboflavin kinaseand functions as prosthetic group of various reactions. FAD (flavinadenine dinucleotide) is a redox cofactor, more specifically aprosthetic group, involved in several important reactions in metabolism.

Thus, in the conversion of 3-methylcrotonic acid into isobutene, in afirst step, a flavin cofactor (FMN or FAD) is modified into a (modified)flavin-derived cofactor. This modification is catalyzed by said FMNprenyl transferase. FMN prenyl transferase prenylates the flavin ring ofthe flavin cofactor (FMN or FAD) into a (modified) prenylated flavincofactor. This reaction is schematically illustrated in FIG. 2A.

In a second step, the actual conversion of 3-methylcrotonic acid intoisobutene is catalyzed by said FMN-dependent decarboxylase via a1,3-dipolar cycloaddition based mechanism wherein said FMN-dependentdecarboxylase uses the prenylated flavin cofactor (FMN or FAD) providedby the associated FMN prenyl transferase. This reaction is schematicallyillustrated in FIG. 2B.

In a preferred embodiment, said FMN prenyl transferase which modifiesthe flavin cofactor (FMN or FAD) into a (modified) flavin-derivedcofactor is a phenylacrylic acid decarboxylase (PAD)-type protein, orthe closely related prokaryotic enzyme UbiX, an enzyme which is involvedin ubiquinone biosynthesis in prokaryotes.

In Escherichia coli, the protein UbiX (also termed3-octaprenyl-4-hydroxybenzoate carboxy-lyase) has been shown to beinvolved in the third step of ubiquinone biosynthesis.

It catalyses the reaction 3-octaprenyl-4-hydroxybenzoate

2-octaprenylphenol+CO₂.

Moreover, the knockout of the homologous protein in yeast (Pad1) hasbeen shown to confer sensitivity to phenylacrylic acid, showing thatthis enzyme functions as a phenylacrylic acid decarboxylase. E. colistrains also contain, in addition to UbiX, a second paralogue namedPad1. Its amino acid sequence shows 52% identity to UbiX and slightlyhigher sequence identity to Saccharomyces cerevisiae phenylacrylic aciddecarboxylase Pad1. Despite its higher sequence similarity with yeastPad1, E. coli Pad1 does not seem to have phenylacrylic aciddecarboxylase activity. Its function is unknown, Pad1 may remove thecarboxylate group from derivatives of benzoic acid but not fromsubstituted phenolic acids.

Thus, in a preferred embodiment, the modification of a flavin cofactor(FMN or FAD) into the corresponding (modified) flavin-derived cofactoris catalyzed by the FMN-containing protein phenylacrylic aciddecarboxylase (PAD). The enzymes involved in the modification of theflavin cofactor (FMN or FAD) into the corresponding modifiedflavin-derived cofactor were initially annotated as decarboxylases (EC4.1.1.-). Some phenylacrylic acid decarboxylases (PAD) are now annotatedas flavin prenyl transferases as EC 2.5.1.-.

In a more preferred embodiment, the conversion of 3-methylcrotonic acidinto isobutene makes use of a phenylacrylic acid decarboxylase(PAD)-type protein as the FMN prenyl transferase which modifies a flavincofactor (FMN or FAD) into the corresponding (modified) flavin-derivedcofactor wherein said phenylacrylic acid decarboxylase (PAD)-typeprotein is derived from Candida albicans (Uniprot accession numberQ5A8L8), Aspergillus niger (Uniprot accession number A3F715),Saccharomyces cerevisiae (Uniprot accession number P33751) orCryptococcus gattii (Uniprot accession number E6R9Z0).

In a preferred embodiment, the phenylacrylic acid decarboxylase(PAD)-type protein employed in the method of the present invention is aphenylacrylic acid decarboxylase (PAD)-type protein derived from Candidaalbicans (Uniprot accession number Q5A8L8; SEQ ID NO:40), Aspergillusniger (Uniprot accession number A3F715; SEQ ID NO:41), Saccharomycescerevisiae (Uniprot accession number P33751; SEQ ID NO:42) orCryptococcus gattii (Uniprot accession number E6R9Z0; SEQ ID NO:43)having the amino acid sequence as shown in SEQ ID NO:40, SEQ ID NO:41,SEQ ID NO:42 and SEQ ID NO:43, respectively.

In a preferred embodiment of the present invention the phenylacrylicacid decarboxylase (PAD)-type protein is an enzyme comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 40 to 43or a sequence which is at least n % identical to any of SEQ ID NOs: 40to 43 with n being an integer between 10 and 100, preferably 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94,95, 96, 97, 98 or 99 and wherein the enzyme has the enzymatic activityof modifying a flavin cofactor (FMN or FAD) into the corresponding(modified) flavin-derived cofactor.

As regards the determination of sequence identity, the following shouldapply: When the sequences which are compared do not have the samelength, the degree of identity either refers to the percentage of aminoacid residues in the shorter sequence which are identical to amino acidresidues in the longer sequence or to the percentage of amino acidresidues in the longer sequence which are identical to amino acidresidues in the shorter sequence. Preferably, it refers to thepercentage of amino acid residues in the shorter sequence which areidentical to amino acid residues in the longer sequence. The degree ofsequence identity can be determined according to methods well known inthe art using preferably suitable computer algorithms such as CLUSTAL.

When using the Clustal analysis method to determine whether a particularsequence is, for instance, at least 60% identical to a referencesequence default settings may be used or the settings are preferably asfollows: Matrix: blosum 30; Open gap penalty: 10.0; Extend gap penalty:0.05; Delay divergent: 40; Gap separation distance: 8 for comparisons ofamino acid sequences. For nucleotide sequence comparisons, the Extendgap penalty is preferably set to 5.0.

In a preferred embodiment ClustalW2 is used for the comparison of aminoacid sequences. In the case of pairwise comparisons/alignments, thefollowing settings are preferably chosen: Protein weight matrix: BLOSUM62; gap open: 10; gap extension: 0.1. In the case of multiplecomparisons/alignments, the following settings are preferably chosen:Protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.2; gapdistance: 5; no end gap.

Preferably, the degree of identity is calculated over the completelength of the sequence.

Amino acid residues located at a position corresponding to a position asindicated herein-below in the amino acid sequence shown in any one ofSEQ ID NOs:40 to 43 can be identified by the skilled person by methodsknown in the art. For example, such amino acid residues can beidentified by aligning the sequence in question with the sequence shownin any one of SEQ ID NOs:40 to 43 and by identifying the positions whichcorrespond to the above indicated positions of any one of SEQ ID NOs:40to 43. The alignment can be done with means and methods known to theskilled person, e.g. by using a known computer algorithm such as theLipman-Pearson method (Science 227 (1985), 1435) or the CLUSTALalgorithm. It is preferred that in such an alignment maximum homology isassigned to conserved amino acid residues present in the amino acidsequences.

In a preferred embodiment ClustalW2 is used for the comparison of aminoacid sequences. In the case of pairwise comparisons/alignments, thefollowing settings are preferably chosen: Protein weight matrix: BLOSUM62; gap open: 10; gap extension: 0.1. In the case of multiplecomparisons/alignments, the following settings are preferably chosen:Protein weight matrix: BLOSUM 62; gap open: 10; gap extension: 0.2; gapdistance: 5; no end gap.

Preferably, the degree of identity is calculated over the completelength of the sequence.

In another preferred embodiment, the modification of a flavin cofactor(FMN or FAD) into the corresponding (modified) flavin-derived cofactoris catalyzed by the FMN-containing protein3-octaprenyl-4-hydroxybenzoate carboxy-lyase also termed UbiX (initiallyannotated EC 4.1.1.-). As mentioned above, the enzymes involved in themodification of the flavin cofactor (FMN or FAD) into the correspondingmodified flavin-derived cofactor were initially annotated asdecarboxylases. Some phenylacrylic acid decarboxylases (PAD) are nowannotated as flavin prenyl transferases as EC 2.5.1.-.

In a more preferred embodiment, the conversion of 3-methylcrotonic acidinto isobutene makes use of a 3-octaprenyl-4-hydroxybenzoatecarboxy-lyase (also termed UbiX) as the FMN prenyl transferase whichmodifies the flavin cofactor (FMN or FAD) into the corresponding(modified) flavin-derived cofactor wherein said3-octaprenyl-4-hydroxybenzoate carboxy-lyase (also termed UbiX) isderived from Escherichia coli (Uniprot accession number POAG03),Bacillus subtilis (Uniprot accession, number A0A086WXG4), Pseudomonasaeruginosa (Uniprot accession number A0A072ZCW8) or Enterobacter sp. DC4(Uniprot accession number W7P6B1).

In an even more preferred embodiment, the 3-octaprenyl-4-hydroxybenzoatecarboxy-lyase (also termed UbiX) employed in the method of the presentinvention is a 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (also termedUbiX) derived from Escherichia coli (Uniprot accession number POAG03;SEQ ID NO:44), Bacillus subtilis (Uniprot accession, number A0A086WXG4;SEQ ID NO:45), Pseudomonas aeruginosa (Uniprot accession numberA0A072ZCW8; SEQ ID NO:46) or Enterobacter sp. DC4 (Uniprot accessionnumber W7P6B1; SEQ ID NO:47) having the amino acid sequence as shown inSEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:47, respectively.

In a preferred embodiment of the present invention the3-octaprenyl-4-hydroxybenzoate carboxy-lyase is an enzyme comprising anamino acid sequence selected from the group consisting of SEQ ID NOs: 44to 47 or a sequence which is at least n % identical to any of SEQ IDNOs: 44 to 47 with n being an integer between 10 and 100, preferably 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92,93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymaticactivity of modifying a flavin cofactor (FMN or FAD) into thecorresponding (modified) flavin-derived cofactor. As regards thedetermination of the sequence identity, the same applies as has been setforth above.

In another preferred embodiment, the modification of a flavin cofactor(FMN or FAD) into the corresponding (modified) flavin-derived cofactoris catalyzed by a flavin prenyl transferase.

As mentioned above, the actual decarboxylation, i.e., the conversion of3-methylcrotonic acid into isobutene is catalyzed by an FMN-dependentdecarboxylase via a 1,3-dipolar cycloaddition based mechanism whereinsaid FMN-dependent decarboxylase uses the prenylated flavin cofactor(FMN or FAD) provided by any of the above described associated FMNprenyl transferases.

In a preferred embodiment, said FMN-dependent decarboxylase catalyzingthe decarboxylation of 3-methylcrotonic acid into isobutene is catalyzedby a ferulic acid decarboxylase (FDC). Ferulic acid decarboxylases (FDC)belong to the enzyme class EC 4.1.1.-.

In an even more preferred embodiment, the conversion of 3-methylcrotonicacid into isobutene makes use of a ferulic acid decarboxylases (FDC)which is derived from Saccharomyces cerevisiae (Uniprot accession numberQ03034), Enterobacter sp. (Uniprot accession number V3P7U0), Bacilluspumilus (Uniprot accession number Q45361), Aspergillus niger (Uniprotaccession number A2R0P7) or Candida dubliniensis (Uniprot accessionnumber B9WJ66).

In a preferred embodiment, the ferulic acid decarboxylases (FDC)employed in the method of the present invention is a ferulic aciddecarboxylases (FDC) derived from Saccharomyces cerevisiae (Uniprotaccession number Q03034; SEQ ID NO:48), Enterobacter sp. (Uniprotaccession number V3P7U0; SEQ ID NO:49), Bacillus pumilus (Uniprotaccession number Q45361; SEQ ID NO:50), Aspergillus niger (Uniprotaccession number A2ROP7; SEQ ID NO:51) or Candida dubliniensis (Uniprotaccession number B9WJ66; SEQ ID NO:52) having the amino acid sequence asshown in SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 and SEQID NO:52, respectively.

In another more preferred embodiment, the conversion of 3-methylcrotonicacid into isobutene makes use of a protocatechuate decarboxylase (EC4.1.1.63).

Thus, in one preferred embodiment, the conversion of 3-methylcrotonicacid into isobutene is catalyzed by a protocatechuate (PCA)decarboxylase (EC 4.1.1.63). PCA decarboxylases (also termed AroY) areknown to catalyze the following reaction, i.e., the enzymatic conversionof protocatechuate (PCA) into catechol (Johnson et al., MetabolicEngineering Communications 3 (2016), 111): 3,4-dihydroxybenzoate

catechol+CO₂

This enzyme occurs in a variety of organisms and has, e.g., beendescribed in Enterobacter aerogenes, Enterobacter cloacae,Rhodopseudomonas sp. and Sedimentibacter hydroxybenzoicus.

In a preferred embodiment of the present invention, the PCAdecarboxylase employed in the method of the present invention is a PCAdecarboxylase which is derived from Klebsiella pneumoniae (Uniprotaccession number B9AM6), Leptolyngbya sp. (Uniprot accession numberA0A0S3U6D8), or Phascolarctobacterium sp. (Uniprot accession numberR611V6).

In a preferred embodiment, the PCA decarboxylase embloyed in the methodof the present invention is an enzyme derived from Klebsiella pneumonia(SEQ ID NO:78), Leptolyngbya sp. (SEQ ID NO:80), orPhascolarctobacterium sp. (SEQ ID NO:81).

In a preferred embodiment of the present invention the PCA decarboxylaseis an enzyme comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 78, 80 and 81 or a sequence which is at leastn % identical to any of SEQ ID NOs: 78, 80 and 81 with n being aninteger between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99and wherein the enzyme has the enzymatic activity of converting3-methylcrotonic acid into isobutene. As regards the determination ofthe sequence identity, the same applies as has been set forth above.

In a preferred embodiment of the present invention the ferulic aciddecarboxylase (FDC) is an enzyme comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 48 to 52 or a sequencewhich is at least n % identical to any of SEQ ID NOs: 48 to 52 with nbeing an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97,98 or 99 and wherein the enzyme has the enzymatic activity of converting3-methylcrotonic acid into isobutene. As regards the determination ofthe sequence identity, the same applies as has been set forth above.

In another preferred embodiment, said FMN-dependent decarboxylasecatalyzing the decarboxylation of 3-methylcrotonic acid into isobuteneis an enzyme which is closely related to the above ferulic aciddecarboxylase (FDC), namely a 3-polyprenyl-4-hydroxybenzoatedecarboxylase (also termed UbiD). 3-polyprenyl-4-hydroxybenzoatedecarboxylase belongs to the UbiD decarboxylase family classified asEC:4.1.1.-.

In a more preferred embodiment, the conversion of 3-methylcrotonic acidinto isobutene makes use of a 3-polyprenyl-4-hydroxybenzoatedecarboxylase (UbiD) which is derived from Hypocrea atroviridis (UniProtAccession number G9NLP8), Sphaerulina musiva (UniProt Accession numberM3DF95), Penecillinum requeforti (UniProt Accession number W6QKP7),Fusarium oxysporum f. sp. lycopersici (UniProt Accession number W9LTH3),Saccharomyces kudriavzevii (UniProt Accession number J8TRN5),Saccaromyces cerevisiae, Aspergillus parasiticus, Candida albicans,Grosmannia clavigera, Escherichia coli (Uniprot accession numberPOAAB4), Bacillus megaterium (Uniprot accession number D5DTL4),Methanothermobacter sp. CaT2 (Uniprot accession number T2GKK5),Mycobacterium chelonae 1518 (Uniprot accession number X8EX86) orEnterobacter cloacae (Uniprot accessin number V3DX94).

In an even more preferred embodiment, the 3-polyprenyl-4-hydroxybenzoatedecarboxylase (UbiD) employed in the method of the present invention isa 3-polyprenyl-4-hydroxybenzoate decarboxylase (UbiD) derived fromEscherichia coli (Uniprot accession number POAAB4; SEQ ID NO:53),Bacillus megaterium (Uniprot accession number D5DTL4; SEQ ID NO:54),Methanothermobacter sp. CaT2 (Uniprot accession number T2GKK5; SEQ IDNO:55) Mycobacterium chelonae 1518 (Uniprot accession number X8EX86; SEQID NO:56), Hypocrea atroviridis (SEQ ID NO:57), Sphaerulina musiva (SEQID NO:58), Penecillinum requeforti (SEQ ID NO:59), Fusarium oxysporum f.sp. lycopersici (SEQ ID NO:60), Saccharomyces kudriavzevii (SEQ IDNO:61), Saccaromyces cerevisiae (SEQ ID NO:62), Aspergillus parasiticus(SEQ ID NO:63), Candida albicans (SEQ ID NO:64), Grosmannia clavigera(SEQ ID NO:65) or Enterobacter cloacae (SEQ ID NO:79) having the aminoacid sequence as shown in SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ IDNO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, and SEQID NO:79, respectively.

In a preferred embodiment of the present invention the3-polyprenyl-4-hydroxybenzoate decarboxylase (UbiD) is an enzymecomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 53 to 65 or a sequence which is at least n % identical toany of SEQ ID NOs: 53 to 65 and SEQ ID NO:79 with n being an integerbetween 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 andwherein the enzyme has the enzymatic activity of converting3-methylcrotonic acid into isobutene. As regards the determination ofthe sequence identity, the same applies as has been set forth above.

As mentioned above, in another aspect, the 3-methylcrotonic aciddecarboxylase may preferably be an aconitate decarboxylase (EC 4.1.1.6).This decarboxylase does not require the association with an FMN prenyltransferase as it has been described for the above decarboxylases and,accordingly, does not require the provision of a prenylated cofactor.

Thus, in one preferred embodiment, the conversion of 3-methylcrotonicacid into isobutene is catalyzed by an aconitate decarboxylase (EC4.1.1.6). Aconitate decarboxylases (EC 4.1.1.6) have been described tocatalyze the following reaction: cis-aconitate

itaconate+CO₂

This enzyme occurs in a variety of organisms, and has, e.g., beendescribed in Aspergillus itaconicus, Aspergillus terreus, Homo sapiensand Mus musculus. In a preferred embodiment, the aconitate decarboxylase(EC 4.1.1.6) employed in the method of the present invention in theconversion of 3-methylcrotonic acid into isobutene is the aconitasedecarboxylase derived from Aspergillus terreus (UniProt accession numberB31UN8), Homo sapiens (UniProt accession number A6NK06) or Mus musculus(UniProt accession number P54987).

In a preferred embodiment, the aconitate decarboxylase (EC 4.1.1.6)employed in the method of the present invention in the conversion of3-methylcrotonic acid into isobutene is a aconitate decarboxylasederived from Aspergillus terreus (SEQ ID NO:66).

In a preferred embodiment of the present invention the aconitatedecarboxylase is an enzyme comprising the amino acid sequence of SEQ IDNO: 66 or a sequence which is at least n % identical to SEQ ID NO: 66with n being an integer between 10 and 100, preferably 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95,96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity ofconverting 3-methylcrotonic acid into isobutene. As regards thedetermination of the sequence identity, the same applies as has been setforth above.

As mentioned above, in another aspect, the 3-methylcrotonic aciddecarboxylase may preferably be a methylcrotonyl-CoA carboxylase (EC6.4.1.4). This decarboxylase does not require the association with anFMN prenyl transferase as it has been described for the abovedecarboxylases and, accordingly, does not require the provision of aprenylated cofactor.

Thus, in one preferred embodiment, the conversion of 3-methylcrotonicacid into isobutene is catalyzed by a methylcrotonyl-CoA carboxylase (EC6.4.1.4). Methylcrotonyl-CoA carboxylases have been described tocatalyze the following reaction:

ATP+3-methylcrotonyl-CoA+HCO₃ ⁻+H⁺

ADP+phosphate+3-methylglutaconyl-CoA, i.e. the carboxylation, but theycan also be used to catalyze the reaction of decarboxylation.Methylcrotonyl-CoA carboxylases occur in a variety of organisms,including eukaryotic and prokaryotic organisms, such as plants, animals,fungi and bacteria. The enzyme has, e.g., been described in Daucuscarota, Glycine max, Hordeum vulgare, Pisum sativum, Solanumlycopersicum, Solanum tuberosum, Zea mays, Arabidopsis sp., Lensculinaris, Homo sapiens, Bos taurus, Rattus norvegicus, Mus musculus,Pagrus major, Emericella nidulans, Pseudomonas aeruginosa, Pseudomonascitronellolis, Pseudomonas amygdali, Acidaminococcus fermentans,Escherichia coli, Mycobacterium sp. and Achromobacter sp.

In a preferred embodiment, the methylcrotonyl-CoA carboxylase (EC6.4.1.4) employed in the method of the present invention in theconversion of 3-methylcrotonic acid into isobutene is amethylcrotonyl-CoA carboxylase derived from Pseudomonas amygdali (SEQ IDNO:67).

In a preferred embodiment of the present invention themethylcrotonyl-CoA carboxylase is an enzyme comprising the amino acidsequence of SEQ ID NO: 67 or a sequence which is at least n % identicalto SEQ ID NO: 67 with n being an integer between 10 and 100, preferably10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91,92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has theenzymatic activity of converting 3-methylcrotonic acid into isobutene.As regards the determination of the sequence identity, the same appliesas has been set forth above.

In another preferred embodiment, the methylcrotonyl-CoA carboxylase (EC6.4.1.4) employed in the method of the present invention in theconversion of 3-methylcrotonic acid into isobutene is amethylcrotonyl-CoA carboxylase derived from Myxcoxoccus xanthus. InMyxococcus xanthus, the the liuB gene codes for an enzyme having the twosubunits AibA and AibB (Li et al., Angew. Chem. Int. Ed. 52 (2013),1304-1308). The methylcrotonyl-CoA carboxylase derived from Myxcoxoccusxanthus is a hetero-dimeric enzyme which are annotated as glutaconyl-CoAtransferase subunits A and B (SEQ ID NOs: 100 and 101).

In a preferred embodiment of the present invention themethylcrotonyl-CoA carboxylase is an enzyme comprising the amino acidsequence of SEQ ID NO: 100 or 101 a sequence which is at least n %identical to SEQ ID NO: 100 or 101 with n being an integer between 10and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 and wherein theenzyme has the enzymatic activity of converting 3-methylcrotonic acidinto isobutene. As regards the determination of the sequence identity,the same applies as has been set forth above.

As mentioned above, in another aspect, the 3-methylcrotonic aciddecarboxylase may preferably be a geranoyl-CoA carboxylase (EC 6.4.1.5).This decarboxylase does not require the association with an FMN prenyltransferase as it has been described for the above decarboxylases and,accordingly, does not require the provision of a prenylated cofactor.

Thus, in another preferred embodiment, the conversion of3-methylcrotonic acid via decarboxylasion into isobutene is catalyzed bya geranoyl-CoA carboxylase (EC 6.4.1.5). Geranoyl-CoA carboxylasesnaturally catalyze the following reaction: ATP+geranoyl-CoA+HCO₃ ⁻+H⁺

ADP+phosphate+3-(4-methylpent-3-en-1-yl) pent-2-enedioyl-CoA

The enzyme occurs in eukaryotes and prokaryotes, such as plants andbacteria. The enzyme has, e.g., been described in Daucus carota, Glycinemax, Zea mays, Pseudomonas sp., Pseudomonas aeruginosa, Pseudomonascitronellolis and Pseudomonas mendocina.

In another aspect, the 3-methylcrotonic acid decarboxylase maypreferably be a 6-methylsalicylate decarboxylase (EC 4.1.1.52).

Thus, in another preferred embodiment, the conversion of3-methylcrotonic acid via decarboxylasion into isobutene is catalyzed bya 6-methylsalicylate decarboxylase (EC 4.1.1.52). 6-methylsalicylatedecarboxylases (EC 4.1.1.52) naturally catalyze the following reaction:

6-methylsalicylate

3-methylphenol+CO₂

The enzyme occurs in a variety of organisms, in particular in eucaryotesand prokaryotes, such as bacteria and fungi. The enzyme has, e.g., beendescribed in Aspergillus clavatus (UniProt Accession number T1PRE6),Penicillium griseofulvum and Valsa friesii.

In a preferred embodiment, the 6-methylsalicylate decarboxylase (EC4.1.1.52) employed in the method of the present invention in theconversion 3-methylcrotonic acid via decarboxylasion into isobutene is a6-methylsalicylate decarboxylase derived from Aspergillus clavatus (SEQID NO:68).

In a preferred embodiment of the present invention the6-methylsalicylate decarboxylase is an enzyme comprising the amino acidsequence of SEQ ID NO: 68 or a sequence which is at least n % identicalto SEQ ID NO: 68 with n being an integer between 10 and 100, preferably10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91,92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme has theenzymatic activity of converting 3-methylcrotonic acid viadecarboxylasion into isobutene. As regards the determination of thesequence identity, the same applies as has been set forth above.

In another aspect, the 3-methylcrotonic acid decarboxylase maypreferably be a 2-oxo-3-hexenedioate decarboxylase (EC 4.1.1.77).

Thus, in another preferred embodiment, the conversion of3-methylcrotonic acid via decarboxylasion into isobutene is catalyzed bya 2-oxo-3-hexenedioate decarboxylase (EC 4.1.1.77). 2-oxo-3-hexenedioatedecarboxylases (EC 4.1.1.77) naturally catalyze the following reaction:

(3E)-2-oxohex-3-enedioate

2-oxopent-4-enoate+CO₂

The enzyme occurs in a variety of organisms, in particular inprokaryotes, such as bacteria. The enzyme has, e.g., been described inBordetella sp., Cupriavidus nexator, Geobacillus stearothermophilus(UniProt Accession number BOVXM8), Pseudomonas putida and Ralstoniapickettii.

In a preferred embodiment, the 2-oxo-3-hexenedioate decarboxylase (EC4.1.1.77) employed in the method of the present invention in theconversion 3-methylcrotonic acid via decarboxylasion into isobutene is a2-oxo-3-hexenedioate decarboxylase derived from Geobacillusstearothermophilus (SEQ ID NO:69).

In a preferred embodiment of the present invention the2-oxo-3-hexenedioate decarboxylase is an enzyme comprising the aminoacid sequence of SEQ ID NO: 69 or a sequence which is at least n %identical to SEQ ID NO: 69 with n being an integer between 10 and 100,preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme hasthe enzymatic activity of converting 3-methylcrotonic acid viadecarboxylasion into isobutene. As regards the determination of thesequence identity, the same applies as has been set forth above.

In another possibility, the 3-methylcrotonic acid decarboxylase maypreferably be a 5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylase (EC4.1.1.68).

Thus, in another preferred embodiment, the conversion of3-methylcrotonic acid via decarboxylasion into isobutene is catalyzed bya 5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylase (EC 4.1.1.68).5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylases (EC 4.1.1.68)naturally catalyze the following reaction:

5-oxopent-3-ene-1,2,5-tricarboxylate

2-oxohept-3-enedioate+CO₂

The enzyme has been described to occur in prokaryotes such as bacteria.The enzyme has, e.g., been described in E. coli and Salmonella dublin.

In a preferred embodiment, the 5-oxopent-3-ene-1,2,5-tricarboxylatedecarboxylase (EC 4.1.1.68) employed in the method of the presentinvention in the conversion 3-methylcrotonic acid via decarboxylasioninto isobutene is a 5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylasederived from Salmonella dublin (SEQ ID NO:70).

In a preferred embodiment of the present invention the5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylase is an enzymecomprising the amino acid sequence of SEQ ID NO: 70 or a sequence whichis at least n % identical to SEQ ID NO: 70 with n being an integerbetween 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 andwherein the enzyme has the enzymatic activity of converting3-methylcrotonic acid via decarboxylasion into isobutene. As regards thedetermination of the sequence identity, the same applies as has been setforth above.

The Enzymatic Conversion of 3-Hydroxyisovalerate (HIV) into3-Methylcrotonic Acid: Step II as Shown in FIG. 1

The 3-methylcrotonic acid which is converted according to the method ofthe present invention into isobutene may itself be provided by anenzymatic reaction.

According to the present invention, the 3-methylcrotonic acid can beprovided via different routes which are schematically shown in FIG. 1.

Thus, according to one option, the 3-methylcrotonic acid may itself beprovided by the enzymatic conversion of 3-hydroxyisovalerate (HIV) into3-methylcrotonic acid. The enzymatic conversion of 3-hydroxyisovalerate(HIV) into 3-methylcrotonic acid (step II as shown in FIG. 1) isschematically illustrated in FIG. 3.

According to the present invention, the enzymatic conversion of3-hydroxyisovalerate (HIV) into said 3-methylcrotonic acid preferablymakes use of an enzyme catalyzing the dehydration of a β-hydroxy acid(i.e., e.g., 3-hydroxyisovalerate (HIV)) into an α,β-unsaturated acid(i.e., e.g., 3-methylcrotonic acid). The term “dehydration” generallyrefers to a reaction involving the removal of H₂O. Enzymes catalyzing3-hydroxyisovalerate (HIV) dehydration are enzymes which catalyze thereaction as shown in FIG. 3. Preferably, such an enzyme belongs to thefamily of hydro-lyases (EC 4.2.-.-).

Preferred examples of such enzymes which are classified as EC 4.2.-.-(i.e., hydro-lyases) are:

-   -   aconitase (EC 4.2.1.3);    -   fumarase (EC 4.2.1.2); and    -   enoyl-CoA hydratase/dehydratease (EC 4.2.1.17).

Thus, in one preferred embodiment, the enzymatic conversion of3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid is achieved by theuse of an aconitase (EC 4.2.1.3). Aconitases (EC 4.2.1.3) (also termedaconitase hydratases) are enzymes which catalyze the following reaction:

Citrate

cis-aconitate+H₂O

The enzyme is known from a variety of organisms, including eukaryoticand prokaryotic organisms, such as plants, animals, fungi and bacteria.The enzyme has, e.g., been described in Acer pseudoplatanus, Advenellakashmirensis, Arabidopsis thaliana, Aspergillus niger, Bacillus cereus,Bacillus subtilis, Bacterioides fragilis, Bos taurus, Caenorhabditiselegans, Citrus elementina, Canis lupus familiaris, Corynebacteriumglutamicum, Drosophila melanogaster, E. coli, Glycine max, Helobacterpylori, Homo sapiens, Mus musculus, Mycobacterium tuberculosis,Nicotiana benthamiana, Plasmodium falciparum, Pseudomonas aeruginosa,Rattus norvegicus, Rattus rattus, Saccharomyces cerevisiae,Saccharomycopsis lipolytica, Salmonella enterica, Sinapis alba,Sinorhizobium meliloti, Solanum tuberosum, Streptomyces aureus,Streptomyces viridochromogenes, Sulfolobus acidocaldarius, Sulfolobussolfataricus, Sus scorfa, Trametes sanguinea, Trypanosoma brucei,Xanthomonas campestris, Xanthomonas euvesicatoria, Yarrowia lipolyticaand Zea mays.

In a preferred embodiment, the aconitase (EC 4.2.1.3) is from Advenellakashmirensis (TrEMBL accession number B3TZE0), Bacterioides fragilis(SwissProt accession number Q8RP87), Caenorhabditis elegans (SwissProtaccession number Q23500), Citrus elementina (UniProt accession numberD3GQLO, D3GQL1, or D3GQL2), Drosophila melanogaster (SwissProt accessionnumber Q9NFX3 or Q9NFX2), E. coli (SwissProt accession number P36683 orUniProt accession number P25516), Homo sapiens (UniProt accession numberP21399 or Q99798), Mus musculus (UniProt accession number P28271),Rattus norvegicus (UniProt accession number Q9ER34 or Q63270), Susscorfa (UniProt accession number P16276) or Trypanosoma brucei(SwissProt accession number Q9NJQ8 or Q9NJQ9).

In a preferred embodiment, the aconitase (EC 4.2.1.3) employed in themethod of the present invention in the conversion of3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid is an aconitasederived from E. coli (SEQ ID NO:71).

In a preferred embodiment of the present invention the aconitase is anenzyme comprising the amino acid sequence of SEQ ID NO: 71 or a sequencewhich is at least n % identical to SEQ ID NO: 71 with n being an integerbetween 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 andwherein the enzyme has the enzymatic activity of converting3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid. As regards thedetermination of the sequence identity, the same applies as has been setforth above.

In another preferred embodiment, the enzymatic conversion of3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid is achieved by theuse of a fumarase (EC 4.2.1.2). Fumarases (EC 4.2.1.2) (also termedfumarase hydratases) are enzymes which catalyze the following reaction:

(S)-malate

fumarate+H₂O

The enzyme is known from a variety of organisms, including eukaryoticand prokaryotic organisms, such as plants, animals, fungi and bacteria.The enzyme has, e.g., been described in Arabidopsis thaliana, Ascarissuum, Azotobacter vinelandii, Brevibacterium flavum, Campylobacter coli,Campylobacter fetus, Campylobacter jejuni, Corynebacterium ammoniagenes,Corynebacterium glutamicum, Erwinia sp., E. coli, Euglena gracilis,Geobacillus stearothermophilus, Gluconacetobacter diazotrophicus,Heliobacter pylori, Homo sapiens, Leishmania major, Mesembryanthemumcrystallinum, Mycobacterium tuberculosis, Pelotomaculumthermopropionicum, Pisum sativum, Pseudomonas aeruginosa, Pseudomonasfluorescens, Pycobaculum neutrophilum, Rattus novegicus, Rhizopusoryzae, Rickettsia prowazekii, Saccharomyces bayanus, Sacchoromycescerevisiae, Solanum lycopersicum, Solanum tuberosum, Streptomycescoelicolor, Streptomyces lividans, Streptomyces thermovulgaris,Sulfolobus solfataricus, Sus scrofa, Thermus sp., Thermus thermophilusand Zea mays.

In a preferred embodiment, the fumarase (EC 4.2.1.2) is from Arabidopsisthaliana (UniProt accession number P93033 or Q9FI53), Ascaris suum(SwissProt accession number Q8NRN8), Corynebacterium glutamicum (UniProtaccession number P28271), E. coli (P05042), Homo sapiens (SwissProtaccession number P07954), Mycobacterium tuberculosis (P9WN93),Pycobaculum neutrophilum (UniProt accession number B1Y931 or B1Y932),Rhizopus oryzae (UniProt accession number P55250), Rickettsia prowazekii(UniProt accession number Q9ZCQ4), Sacchoromyces cerevisiae (SwissProtaccession number P08417), Streptomyces thermovulgaris (SwissProtaccession number A5Y6J1) or Sulfolobus solfataricus (UniProt accessionnumber P39461).

In a preferred embodiment, the fumarase (EC 4.2.1.2) employed in themethod of the present invention in the conversion of3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid is a fumarasederived from E. coli (SEQ ID NO:72).

In a preferred embodiment of the present invention the fumarase is anenzyme comprising the amino acid sequence of SEQ ID NO: 72 or a sequencewhich is at least n % identical to SEQ ID NO: 72 with n being an integerbetween 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 andwherein the enzyme has the enzymatic activity of converting3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid. As regards thedetermination of the sequence identity, the same applies as has been setforth above.

In another preferred embodiment, the enzymatic conversion of3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid is achieved by theuse of an enoyl-CoA hydratase/dehydratase (EC 4.2.1.17). Enoyl-CoAhydratases/dehydratases (EC 4.2.1.17) catalyze the following reaction:

(3S)-3-hydroxyacyl-CoA

trans-2(or 3)-enoyl-CoA+H₂O

Enoyl-CoA hydratase is an enzyme that normally hydrates the double bondbetween the second and third carbon atoms on acyl-CoA. However, it canalso be employed to catalyze the reaction in the reverse direction.

Enoyl-CoA hydratases/dehydratases (EC 4.2.1.17) are also termed3-hydroxyacyl-CoA dehydratases and enoyl-CoA hydratases. Both enzymescatalyze the same reaction while the name of one of these enzymesdenotes one direction of the corresponding reaction while the other namedenotes the reverse reaction. As the reaction is reversible, both enzymenames can be used.

This enzyme, also known as crotonase, is naturally involved inmetabolizing fatty acids to produce both acetyl-CoA and energy. Enzymesbelonging to this class have been described to occur, e.g. in rat(Rattus norvegicus), humans (Homo sapiens), mouse (Mus musculus), wildboar (Sus scrofa), Bos taurus, E. coli, Clostridium acetobutylicum andClostridium aminobutyricum. Nucleotide and/or amino acid sequences forsuch enzymes have been determined, e.g. for rat, humans and Bacillussubtilis and Bacillus anthracis. In principle, any enoyl-CoA hydratase(EC 4.2.1.17) which can catalyze the conversion of 3-hydroxyisovalerate(HIV) into 3-methylcrotonic acid can be used in the context of thepresent invention. In a preferred embodiment the enoyl-CoA hydratase isan enoyl-CoA hydratase of Galactomyces reessii (Dhar et al., J. Ind.Microbiol. Biotechnol. 28 (2002), 81-87), an enoyl-CoA hydratase ofBacillus subtilis (Uniprot G4PBC3; SEQ ID NO: 38) or an enoyl-CoAhydratase of Bacillus anthracis (Uniprot Q81YG6; SEQ ID NO: 39).

In a preferred embodiment, the enoyl-CoA hydratase employed in themethod of the invention has an amino acid sequence as shown in any oneof SEQ ID NOs: 38 or 39 or shows an amino acid sequence which is atleast x% homologous to any one of SEQ ID NOs: 38 or 39 and has theactivity of an enoyl-CoA hydratase with x being an integer between 30and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91,92, 93, 94, 95, 96, 97, 98 or 99 wherein such an enzyme is capable ofconverting 3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid as setforth herein above. As regards the determination of the degree ofidentity, the same applies as has been set forth herein above.

The Enzymatic Condensation of Acetone and Acetyl-CoA into3-Hydroxyisovalerate (HIV): Step III as Shown in FIG. 1

The 3-hydroxyisovalerate (HIV) which is converted according to themethod of the present invention into 3-methylcrotonic acid may itself beprovided by an enzymatic reaction, namely the enzymatic condensation ofacetone and acetyl-CoA into said 3-hydroxyisovalerate (HIV). Thecondensation of acetone and acetyl-CoA into said 3-hydroxyisovalerate(HIV) (step III as shown in FIG. 1) is schematically illustrated in FIG.4.

Thus, the present invention also relates to a method for producingisobutene from acetone in which acetone is first condensed withacetyl-CoA into 3-hydroxyisovalerate (HIV) which is then converted into3-methylcrotonic acid. Further, 3-methylcrotonic acid is then convertedinto isobutene as described herein above.

According to the present invention, the condensation of acetone andacetyl-CoA into 3-hydroxyisovalerate (HIV) preferably makes use of anenzyme which is capable of catalyzing the formation of a covalent bondbetween the carbon atom of the oxo (i.e., the C═O) group of acetone andacetyl-CoA, in particular the methyl group of acetyl-CoA. According tothis reaction scheme, the oxo group of acetone reacts as an electrophileand the methyl group of acetyl-CoA reacts as a nucleophile. The generalreaction of the conversion of acetone and acetyl-CoA is shown in FIG. 4.Enyzmes which are capable of enzymatically condensing acetone andacetyl-CoA into 3-hydroxyisovalerate (HIV) are known in the art andhave, e.g., been described in WO 2011/032934.

Preferably, the enzyme employed in the enzymatic condensation of acetoneand acetyl-CoA into 3-hydroxyisovalerate (HIV) is an enzyme with theactivity of a HMG CoA synthase (EC 2.3.3.10) and/or a PksG proteinand/or an enzyme with the activity of a C-C bond cleavage/condensationlyase, such as a HMG CoA lyase (EC 4.1.3.4). HMG CoA synthase has beendescribed for various organisms.

Examples of HMG CoA synthases from different organisms are given in SEQID NO: 1 to 16. SEQ ID NO: 1 shows the sequence of the cytoplasmic HMGCoA synthase of Caenorhabditis elegans (P54871, gene bank F25B4.6), SEQID NO: 2 shows the sequence of the cytoplasmic HMG CoA synthase ofSchizosaccharomyces pombe (fission yeast; P54874), SEQ ID NO: 3 showsthe sequence of the cytoplasmic HMG CoA synthase of Saccharomycescerevisiae (baker's yeast; P54839, gene bank CAA65437.1), SEQ ID NO: 4shows the sequence of the cytoplasmic HMG CoA synthase of Arabidopsisthaliana (Mouse-ear cress; P54873), SEQ ID NO: 5 shows the sequence ofthe cytoplasmic HMG CoA synthase of Dictyostelium discoideum (Slimemold; P54872, gene bank L2114), SEQ ID NO: 6 shows the sequence of thecytoplasmic HMG CoA synthase of Blattella germanica (German cockroach;P54961, gene bank X73679), SEQ ID NO: 7 shows the sequence of thecytoplasmic HMG CoA synthase of Gallus gallus (Chicken; P23228, genebank CHKHMGCOAS), SEQ ID NO: 8 shows the sequence of the cytoplasmic HMGCoA synthase of Homo sapiens (Human; Q01581, gene bank X66435), SEQ IDNO: 9 shows the sequence of the mitochondrial HMG CoA synthase of Homosapiens (Human; P54868, gene bank X83618), SEQ ID NO: 10 shows thesequence of the mitochondrial HMG CoA synthase of Dictyosteliumdiscoideum (Slime mold; Q86HL5, gene bank XM_638984), SEQ ID NO: 11shows the sequence of the HMG CoA synthase of Staphylococcus epidermidis(Q9FD76), SEQ ID NO: 12 shows the sequence of the HMG CoA synthase ofLactobacillus fermentum (B2GBL1), SEQ ID NO: 13 shows the sequence ofthe HMG CoA synthase of Hyperthermus butylicus (A2BMY8), SEQ ID NO: 14shows the sequence of the HMG CoA synthase of Chloroflexus aggregans(B8G795), SEQ ID NO: 15 shows the sequence of the HMG CoA synthase ofLactobacillus delbrueckii (Q1GAH5) and SEQ ID NO: 16 shows the sequenceof the HMG CoA synthase of Staphylococcus haemolyticus Q4L958 (198>Vdifference compared to wild type protein).

In a preferred embodiment of the present invention the HMG CoA synthaseis an enzyme comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1 to 16 or a sequence which is at least n %identical to any of SEQ ID NOs: 1 to 16 and having the activity of a HMGCoA synthase with n being an integer between 10 and 100, preferably 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92,93, 94, 95, 96, 97, 98 or 99.

As regards the determination of sequence identity, the same applies ashas been set forth above.

Another example for a protein which can be used in the condensation ofacetone and acetyl-CoA into 3-hydroxyisovalerate is a PksG protein. Inthe context of the present application the term “PksG protein” or “aprotein/enzyme having the activity of a PksG protein” refers to anyenzyme which is able to catalyze the reaction which is naturallycatalyzed by the PksG protein, i.e., the transfer of —CH₂COO⁻ fromacetyl-S-AcpK (Ac-S-AcpK) to a β-ketothioester polyketide intermediatelinked to one of the thiolation domains of the PksL protein. This is areaction which is analogous to that catalyzed by HMG CoA synthase withthe difference that the acetyl-thioester of the phosphopantetheyl moietyis attached to a carrier protein rather than to part of Coenzyme A.Although the PksG protein in the reaction which it naturally catalyzestransfers the acetyl group from acetyl-S-AcpK to an acceptor, it hasbeen shown previously that the PksG protein can also effect the reactionwhich is normally catalyzed by HMG CoA synthase, i.e. the synthesis ofHMG CoA starting from acetoacetyl CoA and acetyl CoA.

Examples of PksG proteins are given in SEQ ID NO: 17 and 18. Preferably,the PksG protein is an enzyme comprising an amino acid sequence which isat least n identical to SEQ ID NO: 17 or 18 and having the activity of aPksG protein with n being an integer between 10 and 100, preferably 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92,93, 94, 95, 96, 97, 98 or 99.

SEQ ID NO: 17 shows the amino acid sequence of the PksG protein ofBacillus subtilis (P40830) and SEQ ID NO: 18 shows the amino acidsequence of the PksG protein of Mycobacterium marinum (B2HGT6).

As regards the determination of the degree of sequence identity the sameapplies as has been set forth above in connection with HMG CoA synthase.

Examples of “C—C bond cleavage/condensation lyases” in particularinclude enzymes which are classified as isopropylmalate synthase (EC2.3.3.13), as homocitrate synthase (EC 2.3.3.14) or as4-hydroxy-2-ketovalerate aldolase (EC 4.1.3.39). Isopropylmalatesynthase catalyzes the following reaction:acetyl-CoA+3-methyl-2-oxobutanoate+H₂O

(2S)-2-isopropylmalate+CoA. Examples for such enzymes are thecorresponding enzyme from Brucella abortus (strain 2308; Q2YRT1) and thecorresponding enzyme from Hahella chejuensis (strain KCTC 2396; Q2SFA7).

A homocitrate synthase (EC 2.3.3.14) is an enzyme that catalyzes thechemical reaction acetyl-CoA+H₂O+2-oxoglutarate

(R)-2-hydroxybutane-1,2,4-tricarboxylate+CoA. The4-hydroxy-2-ketovalerate aldolase catalyzes the chemical reaction4-hydroxy-2-oxopentanoate

acetaldehyde+pyruvate.

Examples for enzymes classified as “HMG CoA lyase” or “a protein/enzymehaving the activity of a HMG CoA lyase” in the EC number EC 4.1.3.4, aregiven in SEQ ID NOs: 19 to 25. SEQ ID NO: 19 shows the sequence of theHMG CoA lyase of Zea mays (Accession number B6U7B9, gene bank ACG45252),SEQ ID NO: 20 shows the sequence of the HMG CoA lyase of Danio rerio(Brachydanio rerio; A8WG57, gene bank BC154587), SEQ ID NO: 21 shows thesequence of the HMG CoA lyase of Bos taurus (Uniprot accession numberQ29448) and SEQ ID NO: 22 shows the sequence of the HMG CoA lyase ofHomo sapiens (mitochondrial, Uniprot accession number P35914, gene bankHUMHYMEGLA), SEQ ID NO: 23 shows the sequence of the HMG CoA lyase ofPseudomonas putida (Q88H25), SEQ ID NO: 24 shows the sequence of the HMGCoA lyase of Acinetobacter baumannii (B7H4C6) and SEQ ID NO: 25 showsthe sequence of the HMG CoA lyase of Thermus thermophilus (Q721H0).

In a preferred embodiment of the present invention the HMG CoA lyase isan enzyme comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 19 to 25 or a sequence which is at least n %identical to any of SEQ ID NOs: 19 to 25 and having the activity of aHMG CoA lyase with n being an integer between 10 and 100, preferably 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92,93, 94, 95, 96, 97, 98 or 99.

As regards the determination of the degree of sequence identity the sameapplies as has been set forth above in connection with HMG CoA synthase.

The Enzymatic Conversion of Acetoacetate into Acetone: Step IV as Shownin FIG. 1

The acetone which is converted according to the method of the presentinvention into 3-hydroxyisovalerate (HIV) may itself be provided by anenzymatic reaction, namely the enzymatic conversion of acetoacetate intoacetone. The conversion of acetoacetate into acetone (step IV as shownin FIG. 1) is schematically illustrated in FIG. 5. This reaction is adecarboxylation reaction and is a natural occurring reaction inorganisms capable of producing acetone, i.e., organisms of the genusClostridia.

Thus, the present invention also relates to a method for producingisobutene from acetoacetate in which acetoacetate is first convertedinto acetone which is then condensed with acetyl-CoA into3-hydroxyisovalerate (HIV) which is then converted into 3-methylcrotonicacid as described herein above. Further, said 3-methylcrotonic acid isthen converted into isobutene as described herein above.

According to the present invention, the conversion of acetoacetate intosaid acetone preferably makes use of an acetoacetate decarboxylase (EC4.1.1.4). Nucleotide sequences from several organisms encoding thisenzyme are known in the art, e.g. the adc gene from Clostridiumacetobutylicum (Uniprot accession numbers P23670 and P23673),Clostridium beijerinckii (Clostridium MP; Q9RPK1), Clostridiumpasteurianum (Uniprot accession number P81336), Bradyrhizobium sp.(strain BTAi1/ATCC BAA-1182; Uniprot accession number A5EBU7),Burkholderia mallei (ATCC 10399 A9LBSO), Burkholderia mallei (Uniprotaccession number A3MAE3), Burkholderia mallei FMH A5XJB2, Burkholderiacenocepacia (Uniprot accession number A0B471), Burkholderia ambifaria(Uniprot accession number Q0b5P1), Burkholderia phytofirmans (Uniprotaccession number B2T319), Burkholderia spec. (Uniprot accession numberQ38ZU0), Clostridium botulinum (Uniprot accession number B2TLN8),Ralstonia pickettii (Uniprot accession number B2UIG7), Streptomycesnogalater (Uniprot accession number Q9EYI7), Streptomyces avermitilis(Uniprot accession number Q82NF4), Legionella pneumophila (Uniprotaccession number Q5ZXQ9), Lactobacillus salivarius (Uniprot accessionnumber Q1WVG5), Rhodococcus spec. (Uniprot accession number Q0S7W4),Lactobacillus plantarum (Uniprot accession number Q890G0), Rhizobiumleguminosarum (Uniprot accession number Q1 M911), Lactobacillus casei(Uniprot accession number Q031366), Francisella tularensis (Uniprotaccession number QOBLC9), Saccharopolyspora erythreae (Uniprot accessionnumber A4FKR9), Korarchaeum cryptofilum (Uniprot accession number B1L3N6), Bacillus amyloliquefaciens (Uniprot accession number A7Z8K8),Cochliobolus heterostrophus (Uniprot accession number Q8NJQ3),Sulfolobus islandicus (Uniprot accession number C3ML22) and Francisellatularensis subsp. holarctica (strain OSU18).

In a preferred embodiment, the acetoacetate decarboxylase employed inthe method of the present invention in the conversion of acetoacetateinto acetone is an acetoacetate decarboxylase (EC 4.1.1.4) derived fromClostridium acetobutylicum (Uniprot accession numbers P23670 andP23673).

The Enzymatic Conversion of Acetoacetyl-CoA into Acetoacetate: Step Vaand Step Vb as Shown in FIG. 1

The acetoacetate which is converted according to the method of thepresent invention into acetone may itself be provided by an enzymaticreaction, namely the enzymatic conversion of acetoacetyl-CoA intoacetoacetate. The conversion of acetoacetyl-CoA into acetoacetate can beachieved by two different routes. One possibility is the conversion ofacetoacetyl-CoA into acetoacetate by hydrolysing the CoA thioester ofacetoacetyl-CoA into acetoacetate. This reaction (step Va as shown inFIG. 1) is schematically illustrated in FIG. 6. In another, morepreferred, aspect the CoA group of acetoacetyl-CoA is transferred onacetate, resulting in the formation of acetoacetate and acetyl-CoA. Thisreaction (step Vb as shown in FIG. 1) is schematically illustrated inFIG. 7.

Thus, the present invention also relates to a method for producingisobutene from acetoacetyl-CoA in which acetoacetyl-CoA is firstconverted into acetoacetate which is then converted into acetone whichis then condensed with acetyl-CoA into 3-hydroxyisovalerate (HIV) whichis then converted into 3-methylcrotonic acid as described herein above.Further, said 3-methylcrotonic acid is then converted into isobutene asdescribed herein above.

As mentioned, in one aspect, the CoA thioester of acetoacetyl-CoA ishydrolyzed to result in acetoacetate. According to this aspect of thepresent invention, the enzymatic conversion of acetoacetyl-CoA intoacetoacetate is achieved by preferably making use of an acetoacetyl-CoAhydrolase (EC 3.1.2.11) which naturally catalyzes this reaction.

Acetoacetyl-CoA hydrolases (EC 3.1.2.11) catalyse the followingreaction:

acetoacetyl-CoA+H₂O

CoA+acetoacetate

This enzyme is known from various organisms and has, e.g., beendescribed in eukaryotic organisms. The enzyme has, e.g., been describedin Bos taurus, Columba livia, Gallus gallus, Homo sapiens, Mus musculus,Oncorhynchus mykiss, Oryctolagus cuniculus, or Rattus norvegicus. Thus,in a preferred embodiment, the enzyme is from the genus selected fromthe group consisting of Bos, Columba, Gallus, Mus, Oncorhynchus,Oryctolagus, and Rattus. In a more preferred embodiment, the enzyme isfrom the species selected from the group consisting of Bos taurus,Columba livia, Gallus gallus, Homo sapiens, Mus musculus, Oncorhynchusmykiss, Oryctolagus cuniculus, or Rattus norvegicus. Bos taurus, Columbalivia, Gallus gallus, Homo sapiens, Mus musculus, Oncorhynchus mykiss,Oryctolagus cuniculus, and Rattus norvegicus.

As mentioned, in another, more preferred, possibility, the CoA group ofacetoacetyl-CoA is transferred on acetate, resulting in the formation ofacetoacetate and acetyl-CoA. According to this possibility of thepresent invention, the enzymatic conversion of acetoacetyl-CoA intoacetoacetate is achieved by preferably making use of an enzyme which iscapable of transferring the CoA group of acetoacetyl-CoA on acetate.

Preferably, such an enzyme capable of transferring the CoA group ofacetoacetyl-CoA on acetate belongs to the family of CoA transferases (EC2.8.3.-).

Thus, the present invention relates to a method for the enzymaticconversion of acetoacetyl-CoA into acetoacetate by making use of anenzyme capable of transferring the CoA group of acetoacetyl-CoA onacetate, preferably a CoA transferase (EC 2.8.3.-). A preferred exampleof an enzyme catalysing the conversion of acetoacetyl-CoA intoacetoacetate which can be employed in the method of the presentinvention is an enzyme classified as an acetate CoA transferase (EC2.8.3.8).

Acetate CoA transferases (EC 2.8.3.8) catalyse the following reaction:

acyl-CoA+acetate

a fatty acid anion+acetyl-CoA

Acetate CoA transferases (EC 2.8.3.8) are known from various organisms,e.g., from E. coli in which it is encoded by the atoD gene atoA genes(UniProt accession numbers P76458 and P76459). An acetate CoAtransferase is also known from Clostrtidium acetobutylicum in which itis encoded by the ctfAB gene. Thus, in a preferred embodiment, of theinvention, an acetate CoA transferase (EC 2.8.3.8) is used for theconversion of acetoacetyl-CoA into acetoacetate which is derived from E.coli and which it is encoded by the atoD gene atoA genes (UniProtaccession numbers P76458 and P76459) or which is derived fromClostrtidium acetobutylicum and which it is encoded by the ctfAB gene.

The Enzymatic Conversion of 3-Methylcrotonyl-CoA into 3-MethyicrotonicAcid: Step VI as Shown in FIG. 1

The 3-methylcrotonic acid can be provided by another possible routewhich is described in the following.

Thus, in another embodiment, the 3-methylcrotonic acid which isconverted into isobutene may itself be provided by another enzymaticreaction, namely the enzymatic conversion of 3-methylcrotonyl-CoA into3-methylcrotonic acid. The conversion of 3-methylcrotonyl-CoA into3-methylcrotonic acid (step VI as shown in FIG. 1) is schematicallyillustrated in FIG. 8.

The conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid can,e.g., be achieved in different ways, e.g., by three alternativeenzymatic routes described in the following and as shown in FIG. 1 (stepVIa, step VIb or step VIc as shown in FIG. 1).

Thus, the enzymatic conversion of 3-methylcrotonyl-CoA into3-methylcrotonic acid may be achieved by

-   -   (a) a single enzymatic reaction in which 3-methylcrotonyl-CoA is        directly converted into 3-methylcrotonic acid, preferably by        making use of a CoA transferase (EC 2.8.3.-), preferably a        propionate:acetate-CoA transferase (EC 2.8.3.1), an acetate        CoA-transferase (EC 2.8.3.8) or a succinyl-CoA:acetate        CoA-transferase (EC 2.8.3.18) (step VIa as shown in FIG. 1);    -   (b) a single enzymatic reaction in which 3-methylcrotonyl-CoA is        directly converted into 3-methylcrotonic acid, preferably by        making use of a thioester hydrolase (EC 3.1.2.-), preferably an        acetyl-CoA hydrolase (EC 3.1.2.1), an ADP-dependent        short-chain-acyl-CoA hydrolase (EC 3.1.2.18) or an acyl-CoA        hydrolase (EC 3.1.2.20) (step VIb as shown in FIG. 1); or    -   (c) two enzymatic steps comprising        -   (i) first enzymatically converting 3-methylcrotonyl-CoA into            3-methylcrotonyl phosphate; and        -   (ii) then enzymatically converting the thus obtained            3-methylcrotonyl phosphate into said 3-methylcrotonic acid            (step VIc as shown in FIG. 1).

Thus, one possibility is a two-step conversion from 3-methylcrotonyl-CoAvia 3-methylcrotonyl phosphate into 3-methylcrotonic acid. Two otheroptions involve a direct conversion of 3-methylcrotonyl-CoA into3-methylcrotonic acid. These three options will be discussed in thefollowing.

Accordingly, in one embodiment, the enzymatic conversion of3-methylcrotonyl-CoA into 3-methylcrotonic acid is achieved by twoenzymatic steps comprising (i) first enzymatically converting3-methylcrotonyl-CoA into 3-methylcrotonyl phosphate; and (ii) thenenzymatically converting the thus obtained 3-methylcrotonyl phosphateinto said 3-methylcrotonic acid (as shown in step VIc of FIG. 1). Thecorresponding reaction is schematically shown in FIG. 11.

The conversion of 3-methylcrotonyl-CoA into 3-methylcrotonyl phosphatecan, e.g., be achieved by the use of a phosphate butyryltransferase (EC2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8).

Phosphate butyryltransferase (EC 2.3.1.19) naturally catalyzes thefollowing reaction Butyryl-CoA+H₃PO₄

butyryl phosphate+CoA

It has been described by Wiesenborn et al. (Appl. Environ. Microbiol. 55(1989), 317-322) and by Ward et al. (J. Bacteriol. 181 (1999),5433-5442) that phosphate butyryltransferases (EC 2.3.1.19) can use anumber of substrates in addition to butyryl coenzyme A (butyryl-CoA), inparticular acetyl-CoA, propionyl-CoA, isobutyryl-CoA, valeryl-CoA andisovaleryl-CoA.

The enzyme has been described to occur in a number of organisms, inparticular in bacteria and in protozoae. In one embodiment the enzyme isfrom the protozoae Dasytricha ruminantium. In a preferred embodiment thephosphate butyryltransferase is a phosphate butyryltransferase from abacterium, preferably from a bacterium of the genus Bacillus,Butyrivibrio, Enterococcus or Clostridium, more preferably Enterococcusor Clostridium, and even more preferably from Bacillus megaterium,Bacillus subtilis, Butyrivibrio fibrisolvens, Clostridiumacetobutylicum, Clostridium beijerinckii, Clostridium butyricum,Clostridium kluyveri, Clostridium saccharoacetobutylicum, Clostridiumsprorogenes or Enterococcus faecalis. Most preferably, the enzyme isfrom Clostridium acetobutylicum, in particular the enzyme encoded by theptb gene (Uniprot Accession number F0K6W0; Wiesenborn et al. (Appl.Environ. Microbiol. 55 (1989), 317-322)) or from Enterococcus faecalis(Uniprot Accession number K4YRE8; Ward et al. (J. Bacteriol. 181 (1999),5433-5442)).

In a preferred embodiment, the conversion of 3-methylcrotonyl-CoA into3-methylcrotonyl phosphate is achieved by making use of a phosphatebutyryltransferase from Clostridium acetobutylicum, preferably fromClostridium acetobutylicum strain ATCC 824. The amino acid sequence ofsaid protein is shown in SEQ ID NO: 26.

It is, of course, not only possible to use an enzyme exactly showingthis amino acid of SEQ ID NO:26. It is also possible to use an enzymewhich comprises a sequence which is at least 60% identical to the aminoacid sequence shown in SEQ ID NO: 26. Preferably, the sequence identityis at least 70%, more preferably at least 80%, 85% or 90%, even morepreferably 91%, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularlypreferred at least 99% to SEQ ID NO:26 and the enzyme has the enzymaticactivity of converting 3-methylcrotonyl-CoA into 3-methylcrotonylphosphate. As regards the determination of the sequence identity, thesame applies as has been set forth above.

In another preferred embodiment, the conversion of 3-methylcrotonyl-CoAinto 3-methylcrotonyl phosphate is achieved by making use of a phosphatebutyryltransferase from Bacillus subtilis, preferably from Bacillussubtilis having the UniProt Accession number P54530. The amino acidsequence of said protein is shown in SEQ ID NO: 73.

In a preferred embodiment of the present invention the phosphatebutyryltransferase is an enzyme comprising the amino acid sequence ofSEQ ID NO: 73 or a sequence which is at least n % identical to SEQ IDNO: 73 with n being an integer between 10 and 100, preferably 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93,94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymaticactivity of converting 3-methylcrotonyl-CoA into 3-methylcrotonylphosphate. As regards the determination of the sequence identity, thesame applies as has been set forth above.

In another preferred embodiment, the conversion of 3-methylcrotonyl-CoAinto 3-methylcrotonyl phosphate is achieved by making use of a phosphatebutyryltransferase from Enterococcus faecalis, preferably fromEnterococcus faecalis having the UniProt Accession number S4BZL5. Theamino acid sequence of said protein is shown in SEQ ID NO: 74.

In a preferred embodiment of the present invention the phosphatebutyryltransferase is an enzyme comprising the amino acid sequence ofSEQ ID NO: 74 or a sequence which is at least n % identical to SEQ IDNO: 74 with n being an integer between 10 and 100, preferably 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93,94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymaticactivity of converting 3-methylcrotonyl-CoA into 3-methylcrotonylphosphate. As regards the determination of the sequence identity, thesame applies as has been set forth above.

Phosphate acetyltransferase (EC 2.3.1.8) naturally catalyzes thefollowing reaction

Acetyl-CoA+H₃PO₄

acetyl phosphate+CoA

It has been described by Veit et al. (J. Biotechno1.140 (2009), 75-83)that phosphate acetyltransferase can also use as a substrate butyryl-CoAor propionyl-CoA.

The accession numbers for this enzyme family in InterPro database areIPR012147 and IPR002505, “http://www.ebi.ac.uk/interpro/entry/IPR002505”

(http://www.ebi.ac.uk/interpro/entry/IPR012147

http://www.ebi.ac.uk/interpro/entry/IPR002505)

See also http://pfam.sanger.ac.uk/family/PF01515

The enzyme has been described in a variety of organisms, in particularbacteria and fungi. Thus, in one preferred embodiment the enzyme is anenzyme from a bacterium, preferably of the genus Escherichia,Chlorogonium, Clostridium, Veillonella, Methanosarcina, Corynebacterium,Ruegeria, Salmonella, Azotobacter, Bradorhizobium, Lactobacillus,Moorella, Rhodopseudomonas, Sinorhizobium, Streptococcus, Thermotoga orBacillus, more preferably of the species Escherichia coli, Chlorogoniumelongatum, Clostridium kluyveri, Clostridium acetobutylicum, Clostridiumacidurici, Veillonella parvula, Methanosarcina thermophila,Corynebacterium glutamicum, Ruegeria pomeroyi, Salmonella enterica,Azotobacter vinelandii, Bradyrhizobium japonicum, Lactobacillusfermentum, Lactobacillus sanfranciscensis, Moorella thermoacetica,Rhodopseudomonas palustris, Sinorhizobium meliloti, Streptococcuspyogenes, Thermotoga maritima or Bacillus subtilis. In another preferredembodiment the enzyme is an enzyme from a fungus, preferably from thegenus Saccharomyces, more preferably of the species Saccharomycescerevisiae.

In a preferred embodiment, the conversion of 3-methylcrotonyl-CoA into3-methylcrotonyl phosphate is achieved by making use a phosphateacetyltransferase from Corynebacterium glutamicum, preferably fromCorynebacterium glutamicum strain ATCC 13032. The amino acid sequence ofsaid protein is shown in SEQ ID NO: 27.

It is, of course, not only possible to use an enzyme exactly showingthis amino acid of SEQ ID NO:27. It is also possible to use an enzymewhich comprises a sequence which is at least 60% identical to the aminoacid sequence shown in SEQ ID NO: 27. Preferably, the sequence identityis at least 70%, more preferably at least 80%, 85% or 90%, even morepreferably 91%, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularlypreferred at least 99% to SEQ ID NO:27 and the enzyme has the enzymaticactivity of converting 3-methylcrotonyl-CoA into 3-methylcrotonylphosphate. As regards the determination of the sequence identity, thesame applies as has been set forth above.

The conversion of 3-methylcrotonyl phosphate into 3-methylcrotonic acidcan, e.g., be achieved by making use of an enzyme which is classified asEC 2.7.2.-, i.e., a phosphotransferase. Such enzymes use a carboxy groupas acceptor. Thus, the conversion of 3-methylcrotonyl phosphate into3-methylcrotonic acid can, e.g., be achieved by making use of an enzymewith a carboxy group as acceptor (EC 2.7.2.-). In a preferredembodiment, the conversion of 3-methylcrotonyl phosphate into3-methylcrotonic acid is achieved by the use of a propionate kinase (EC2.7.2.15), an acetate kinase (EC 2.7.2.1), a butyrate kinase (EC2.7.2.7) or a branched-chain-fatty-acid kinase (EC 2.7.2.14).

Butyrate kinases (EC 2.7.2.7) naturally catalyze the following reaction

Butyrate+ATP

butyryl phosphate+ADP

It has been described, e.g. by Hartmanis (J. Biol. Chem. 262 (1987),617-621) that butyrate kinase can use a number of substrates in additionto butyrate, e.g. valerate, isobutyrate, isovalerate and vinyl acetate.The enzyme has been described in a variety of organisms, in particularbacteria. In one preferred embodiment the enzyme is from a bacterium,preferably from a bacterium of the genus Clostridium, Butyrivibrio,Thermotoga or Enterococcus. Preferred is Clostridium. More preferablythe enzyme is from a bacterium of the species Clostridiumacetobutylicum, Clostridium proteoclasticum, Clostridium tyrobutyricum,Clostridium butyricum, Clostridium pasteurianum, Clostridiumtetanomorphum, Butyrivibrio firbrosolvens, Butyrivibrio hungatei,Thermotoga maritime or Enterococcus durans. Preferred is Clostridiumacetobutylicum. For this organism two butyrate kinases have beendescribed: butyrate kinase 1 (Uniprot Accession number: Q45829) andbutyrate kinase II (Uniprot Accession number: 0971119).

In another preferred embodiment, the conversion of 3-methylcrotonylphosphate into 3-methylcrotonic acid is achieved by making use of abutyrate kinase from Lactobacillus, preferably from Lactobacillus casei(UniProt Accession number K0N529) or a butyrate kinase from Geobacillus,preferably from Geobacillus sp. (UniProt Accession number L8A0E1). Theamino acid sequence of these proteins are shown in SEQ ID NO:75 and SEQID NO:76, respectively.

In a preferred embodiment of the present invention the butyrate kinaseis an enzyme comprising the amino acid sequence of SEQ ID NO: 75 or 76or a sequence which is at least n % identical to SEQ ID NO: 75 or 76with n being an integer between 10 and 100, preferably 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95,96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity ofconverting 3-methylcrotonyl phosphate into 3-methylcrotonic acid. Asregards the determination of the sequence identity, the same applies ashas been set forth above.

Branched-chain-fatty-acid kinases (EC 2.7.2.14) naturally catalyze thefollowing reaction

Alkyl carboxylic acid+ATP

acyl phosphate+ADP

wherein “alkyl” may be 2-methylbutanoate, butanoate, isobutanoate,pentanoate or propionate. The latter reaction with propionate has beendescribed for a branched-chain fatty acid kinase from a spirochaete (J.Bacteriol. 152 (1982), 246-54).

This enzyme has been described to occur in a number of bacteria. Thus,in one preferred embodiment the enzyme is an enzyme from a bacterium,preferably of the genus Spirochaeta or Thermotoga, more preferablyThermotoga maritime.

Propionate kinases (EC 2.7.2.15) naturally catalyze the followingreactions

Propanoate+ATP

propanoyl phosphate+ADP

Acetate+ATP

acetyl phosphate+ADP

This enzyme has been described to occur in a number of bacteria, inparticular Enterobacteriacea. Thus, in one preferred embodiment theenzyme is an enzyme from a bacterium, preferably of the genus Salmonellaor Escherichia, more preferably of the species Salmonella enterica,Salmonella typhimurium or Escherichia coli.

In a preferred embodiment, the conversion of 3-methylcrotonyl phosphateinto 3-methylcrotonic acid is achieved by making use of a propionatekinase from Salmonella typhimurium, preferably from Salmonellatyphimurium strain ATCC 700720. The amino acid sequence of said proteinis shown in SEQ ID NO: 28.

It is, of course, not only possible to use an enzyme exactly showingthis amino acid of SEQ ID NO:28. It is also possible to use an enzymewhich comprises a sequence which is at least 60% identical to the aminoacid sequence shown in SEQ ID NO: 28. Preferably, the sequence identityis at least 70%, more preferably at least 80%, 85% or 90%, even morepreferably 91%, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularlypreferred at least 99% to SEQ ID NO:28 and the enzyme has the enzymaticactivity of converting 3-methylcrotonyl phosphate into 3-methylcrotonicacid. As regards the determination of the sequence identity, the sameapplies as has been set forth above.

In another preferred embodiment, the conversion of 3-methylcrotonylphosphate into 3-methylcrotonic acid is achieved by making use of apropionate kinase from Escherichia coli, preferably from Escherichiacoli strain K12. The amino acid sequence of said protein is shown in SEQID NO: 29.

It is, of course, not only possible to use an enzyme exactly showingthis amino acid of SEQ ID NO:29. It is also possible to use an enzymewhich comprises a sequence which is at least 60% identical to the aminoacid sequence shown in SEQ ID NO: 29. Preferably, the sequence identityis at least 70%, more preferably at least 80%, 85% or 90%, even morepreferably 91%, 92%, 93,%, 94%, 95%, 96%, 97%, 98% and particularlypreferred at least 99% to SEQ ID NO:29 and the enzyme has the enzymaticactivity of converting 3-methylcrotonyl phosphate into 3-methylcrotonicacid. As regards the determination of the sequence identity, the sameapplies as has been set forth above.

Acetate kinases (EC 2.7.2.1) naturally catalyze the following reaction

Acetate+ATP

acetyl phosphate+ADP

This enzyme has been described to occur in a number of organisms, inparticular bacteria and eukaryotes. In one preferred embodiment theenzyme is from a bacterium, preferably from a bacterium of the genusMethanosarcina, Cryptococcus, Ethanoligenens, Propionibacterium,Roseovarius, Streptococcus, Salmonella, Acholeplasma, Acinetobacter,Ajellomyces, Bacillus, Borrelia, Chaetomium, Clostridium, Coccidioides,Coprinopsis, Cryptococcus, Cupriavidus, Desulfovibrio, Enterococcus,Escherichia, Ethanoligenes, Geobacillus, Helicobacter, Lactobacillus,Lactococcus, Listeria, Mesoplasma, Moorella, Mycoplasma, Oceanobacillus,Propionibacterium, Rhodospeudomonas, Roseovarius, Salmonella,Staphylococcus, Thermotoga or Veillonella, more preferably from abacterium of the species Methanosarcina thermophila, Cryptococcusneoformans, Ethanoligenens harbinense, Propionibacteriumacidipropionici, Streptococcus pneumoniae, Streptococcus enterica,Streptococcus pyogenes, Acholeplasma laidlawii, Acinetobactercalcoaceticus, Ajellomyces capsulatus, Bacillus subtilis, Borreliaburgdorferi, Chaetomium globosum, Clostridium acetobutylicum,Clostridium thermocellum, Coccidioides immitis, Coprinopsis cinerea,Cryptococcus neoformans, Cupriavidus necator, Desulfovibrio vulgaris,Enterococcus faecalis, Escherichia coli, Ethanoligenes harbinense,Geobacillus stearothermophilus, Helicobacter pylori, Lactobacillusdelbrueckii, Lactobacillus acidophilus, Lactobacillus sanfranciscensis,Lactococcus lactis, Listeria monocytogenes, Mesoplasma florum,Methanosarcina acetivorans, Methanosarcina mazei, Moorellathermoacetica, Mycoplasma pneumoniae, Oceanobacillus iheyensis,Propionibacterium freudenreichii, Propionibacterium acidipropionici,Rhodospeudomonas palustris, Salmonella enteric, Staphylococcus aureus,Thermotoga maritime or Veillonella parvula.

In another preferred embodiment the enzyme is an enzyme from a fungus,preferably from a fungus of the genus Aspergillus, Gibberella, Hypocrea,Magnaporthe, Phaeosphaeria, Phanerochaete, Phytophthora, Sclerotinia,Uncinocarpus, Ustilago or Neurospora even more preferably from a fungusof the species Aspergillus fumigates, Aspergillus nidulans, Gibberellazeae, Hypocrea jecorina, Magnaporthe grisea, Phaeosphaeria nodorum,Phanerochaete chrysosporium, Phytophthora ramorum, Phytophthora sojae,Sclerotinia sclerotiorum, Uncinocarpus reesii, Ustilago maydis orNeurospora crassa.

In a further preferred embodiment the enzyme is an enzyme from a plantor an algae, preferably from the genus Chlamydomonas, even morepreferably from the species Chlamydomonas reinhardtii.

In another embodiment the enzyme is from an organism of the genusEntamoeba, more preferably of the species Entamoeba histolytica.

The above mentioned enzyme families suitable for the conversion of3-methylcrotonyl-CoA into 3-methylcrotonyl phosphate have been shown tobe evolutionary related and contain common sequence signatures. Thesessignatures are referenced and described in Prosite database:

http://prosite.expasy.org/cgi-bin/prosite/nicedoc.pl?PS01075

Gao et al. (FEMS Microbiol. Lett. 213 (2002), 59-65) already describedgenetically modified E. coli cells which have been transformed, interalia, with the ptb gene and the buk gene from Clostridium acetobutylicumencoding a phosphate butyryltransferase (EC 2.3.1.19) and a butyratekinase (EC 2.7.2.7), respectively. These E. coli cells have been shownto be able to produce D-(−)-3-hydroxybutyric acid (3HB).

As mentioned above, the conversion of 3-methylcrotonyl-CoA into3-methylcrotonic acid can also be achieved by two alternativeconversions wherein 3-methylcrotonyl-CoA is directly converted into3-methylcrotonic acid.

Preferably, in one embodiment, 3-methylcrotonyl-CoA is directlyconverted into 3-methylcrotonic acid by hydrolyzing the thioester bondof 3-methylcrotonyl-CoA into 3-methylcrotonic acid by making use of anenzyme which belongs to the family of thioester hydrolases (in thefollowing referred to as thioesterases (EC 3.1.2.-)). This reaction isschematically shown in FIG. 10.

Examples for preferred thioester hydrolases (EC 3.1.2.-) are anacetyl-CoA hydrolase (EC 3.1.2.1), an ADP-dependent short-chain-acyl-CoAhydrolase (EC 3.1.2.18) and an acyl-CoA hydrolase (EC 3.1.2.20) (stepVIb as shown in FIG. 1).

In an alternative embodiment, 3-methylcrotonyl-CoA is directly convertedinto 3-methylcrotonic acid, preferably by making use of an enzyme whichbelongs to the family of CoA-transferases (EC 2.8.3.-). This reaction isschematically shown in FIG. 9.

Examples for preferred CoA transferases (EC 2.8.3.-) are apropionate:acetate-CoA transferase (EC 2.8.3.1), an acetateCoA-transferase (EC 2.8.3.8) and a succinyl-CoA:acetate CoA-transferase(EC 2.8.3.18) (step VIa as shown in FIG. 1).

Thioesterases (TEs; also referred to as thioester hydrolases) areenzymes which are classified as EC 3.1.2. Presently thioesterases areclassified as EC 3.1.2.1 through EC 3.1.2.30 while TEs which are not yetclassified/unclassified are grouped as enzymes belonging to EC 3.1.2.-.Cantu et al. (Protein Science 19 (2010), 1281-1295) describe that thereare 23 families of thioesterases which are unrelated to each other asregards the primary structure. However, it is assumed that all membersof the same family have essentially the same tertiary structure.Thioesterases hydrolyze the thioester bond between a carbonyl group anda sulfur atom.

In a preferred embodiment, a thioesterase employed in a method accordingto the present invention for converting 3-methylcrotonyl-CoA into3-methylcrotonic acid is selected from the group consisting of:

-   -   acetyl-CoA hydrolase (EC 3.1.2.1);    -   palmitoyl-CoA hydrolase (EC 3.1.2.2);    -   3-hydroxyisobutyryl-CoA hydrolase (EC 3.1.2.4);    -   oleoyl-[acyl-carrier-protein] hydrolase (EC 3.1.2.14);    -   ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18);    -   ADP-dependent medium-chain-acyl-CoA hydrolase (EC 3.1.2.19); and    -   acyl-CoA hydrolase (EC 3.1.2.20).

Thus, in one preferred embodiment the direct conversion of3-methylcrotonyl-CoA into 3-methylcrotonic acid is achieved by makinguse of an acetyl-CoA hydrolase (EC 3.1.2.1). Acetyl-CoA hydrolases areenzymes which catalyze the following reaction:

Acetyl-CoA+H₂O→acetate+CoA

This enzyme occurs in a variety of organism, including eukaryotic andprokaryotic organisms, such as plants, animals, fungi and bacteria. Theenzyme has, e.g., been described in Rattus norvegicus (Uniprot Accessionnumber: Q99NB7), Mus musculus, Sus scrofa, Bos taurus, Gallus gallus,Platyrrhini, Ovis aries, Mesocricetus auratus, Ascaris suum, Homosapiens (Uniprot Accession number: Q8WYKO), Pisum sativum, Cucumissativus, Panicus sp., Ricinus communis, Solanum tuberosum, Spinaciaoleracea, Zea mays, Glycine max, Saccharomyces cerevisiae, Neurosporacrassa, Candida albicans, Trypanosoma brucei brucei, Trypanosoma cruzi,Trypanosoma dionisii, Trypanosoma vespertilionis, Crithidia fasciculate,Clostridium aminovalericum, Acidaminococcus fermaentans, Bradyrhizobiumjaponicum and Methanosarcina barkeri.

In another preferred embodiment the direct conversion of3-methylcrotonyl-CoA into 3-methylcrotonic acid is achieved by makinguse of a palmitoyl-CoA hydrolase (EC 3.1.2.2). Palmitoyl-CoA hydrolasesare enzymes which catalyze the following reaction:

Palmitoyl-CoA+H₂O→palmitate+CoA

This enzyme occurs in a variety of organism, including eukaryotic andprokaryotic organisms, such as plants, animals, fungi and bacteria. Theenzyme has, e.g., been described in Arabidopsis thaliana (UniprotAccession number: Q8GYW7), Pisum sativum, Spinacia oleracea,Bumilleriopsis filiformis, Eremosphaera viridis, Mougeotia scalaris,Euglena gracilis, Rhodotorula aurantiaca, Saccharaomyces cerevisiae,Candida rugosa, Caenorhabditis elegans, Mus musculus (Uniprot Accessionnumber: P58137), Homo sapiens, Platyrrhini, Bos taurus, Canis lupusfamiliaris, Sus scrofa, Cavia procellus, Columba sp., Cricetulusgriseus, Mesocricetus auratus, Drosophila melanogaster, Rattusnorvegicus, Oryctolagus cuniculus, Gallus gallus, Anas platyrhynchos,Mycobacterium tuberculosis, Mycobacterium phlei, Mycobacteriumsmegmatis, Acinetobacter colcaceticus, Haemophilus influenza,Helicobacter pylori, Bacillus subtilis, Pseudomonas aeruginosa,Rhodobacter shpaeroides, Streptomyces coelicolor, Streptomycesvenezuelae and E. coli.

In a further preferred embodiment the direct conversion of3-methylcrotonyl-CoA into 3-methylcrotonic acid is achieved by makinguse of a 3-hydroxyisobutyryl-CoA hydrolase (EC 3.1.2.4).3-hydroxyisobutyryl-CoA hydrolases are enzymes which catalyze thefollowing reaction:

3-hydroxyisobutyryl-CoA+H₂O→3-hydroxyisobutyrate+CoA

This enzyme occurs in a variety of organism, including eukaryotic andprokaryotic organisms, such as plants, animals, fungi and bacteria. Theenzyme has, e.g., been described in Arabidopsis thaliana, Homo sapiens,Canus lupus familiaris, Rattus norvegicus, Bacillus cereus, Pseudomonasfluorescens and Pseudomonas aeruginosa.

In yet another preferred embodiment the direct conversion of3-methylcrotonyl-CoA into 3-methylcrotonic acid is achieved by makinguse of an oleoyl-[acyl-carrier-protein] hydrolase (EC 3.1.2.14).Oleoyl-[acyl-carrier-protein] hydrolases are enzymes which catalyze thefollowing reaction:

oleoyl-[acyl-carrier-protein]+H₂O→oleate+[acyl-carrier-protein]

This enzyme occurs in a variety of plants and bacteria. The enzyme has,e.g., been described in Arabidopsis thaliana, Allium ampeloprasum,Curcurbita moschata, Cuphea calophylla, Cuphea hookeriana, Cuphealanceolata, Cuphea wrightii, Umbellularia californica, Coriandrumsativum, Spinacia oleracea, Elaeis sp., Elaeis guineensis, Glycine max,Persea americana, Pisum sativum, Sinapis alba, Ulmus americana, Zeamays, Brassica juncea, Brassica napus, Brassica rapa subsp. campestris,Jatropha curcas, Ricinus communis, Cinnamomum camphorum, Macadamiatetraphylla, Magnifera indica, Madhuca longifolia, Populus tomentosa,Chimonanthus praecox, Gossypium hirsutum, Diploknema butyracea,Helianthus annuus and Streptococcus pyogenes.

In yet another preferred embodiment the direct conversion of3-methylcrotonyl-CoA into 3-methylcrotonic acid is achieved by makinguse of an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18).ADP-dependent short-chain-acyl-CoA hydrolases are enzymes which catalyzethe following reaction:

an acyl-CoA+H₂O a carboxylate+CoA

This enzyme occurs in a variety of animals and has, e.g., been describedin Mus musculus, Rattus norvegicus and Mesocricetus auratus.

In yet another preferred embodiment the direct conversion of3-methylcrotonyl-CoA into 3-methylcrotonic acid is achieved by makinguse of an ADP-dependent medium-chain-acyl-CoA hydrolase (EC 3.1.2.19).ADP-dependent medium-chain-acyl-CoA hydrolases are enzymes whichcatalyze the following reaction:

an acyl-CoA+H₂O→a carboxylate+CoA

This enzyme occurs in a variety of animals and has, e.g., been describedin Rattus norvegicus and Mesocricetus auratus.

In a further preferred embodiment the direct conversion of3-methylcrotonyl-CoA into 3-methylcrotonic acid is achieved by makinguse of an acyl-CoA hydrolase (EC 3.1.2.20). Acyl-CoA hydrolases areenzymes which catalyze the following reaction:

an acyl-CoA+H₂O→a carboxylate+CoA

This enzyme occurs in a variety of organism, including eukaryotic andprokaryotic organisms, such as plants, animals, fungi and bacteria. Theenzyme has, e.g., been described in Arabidopsis thaliana, Rhodotorulaaurantiaca, Bumilleriopsis filiformis, Eremosphaera viridis, Euglenagracilis, Mus musculus, Rattus norvegicus, Homo sapiens, Sus, scrofa,Bos taurus, Cais lupus familiaris, Cavia porcellus, Cricetus griseus,Drosophila melanogaster, Anas platyrhynchos, Gallus gallus,Caenorhabditis elegans, Saccharomyces cerevisia, Candida rugosa,Escherichia coli, Haemophilus influenzae, Xanthomonas campestris,Streptomyces sp., Streptomyces coelicolor, Alcaligenes faecalis,Pseudomonas aeruginosa, Pseudomonas putida, Amycolatopsis mediterranei,Acinetobacter calcoaceticus, Helicobacter pylori, Rhodobacter spaeroidesand Mycobacterium phlei. In a preferred embodiment the acyl-CoAhydrolase is an enzyme from Escherichia coli, from Pseudomonas putida orfrom Haemophilus influenza, more preferably the YciA enzyme from E. colior its closely related homolog H10827 from Haemophilus influenza (Zhuanget al., Biochemistry 47 (2008), 2789-2796). The YciA enzyme fromHaemophilus influenza is described to catalyze the hydrolysis ofpropionyl-CoA into propionic acid (Zhuang et al., Biochemistry 47(2008), 2789-2796). In another preferred embodiment the acetyl-CoAhydrolase is an enzyme from Homo sapiens (UniProt: Q9NPJ3) which isdescribed to hydrolyze propionyl-CoA (Cao et al., Biochemistry 48(2009), 1293-1304).

Particularly preferred enzymes are the above-described acyl-CoAhydrolase YciA enzyme from Haemophilus influenza strain R2866 (SEQ IDNO: 30) and the acetyl-CoA hydrolase enzyme from Homo sapiens (UniProt:Q9NPJ3; SEQ ID NO:31). Particularly preferred are also the enzymesacyl-CoA thioester hydrolase from E. coli (Uniprot POA8ZO; SEQ ID NO:32), acyl-CoA thioesterase 2 from E. coli (Uniprot POAGG2; SEQ ID NO:33) and acyl-CoA thioesterase II from Pseudomonas putida (UniprotQ88DR1; SEQ ID NO: 34). Particularly preferred is the thioesterase TesBfrom E. coli K12 (uniprot :POAGG2), as this enzyme is already describedto efficiently catalyze this reaction in E. coli for the biosynthesis ofpropionic acid (Tseng and Prather, P.N.A.S. 2012, 109(44),p17925-17930).

In another preferred embodiment, the acyl-CoA hydrolase is an enzymederived from the family of 1,4-dihydroxy-2-naphthoyl-CoA hydrolases.Enzymes of this family of 1,4-dihydroxy-2-naphthoyl-CoA hydrolases areknown to catalyze the following reaction:

1,4-dihydroxy-2-naphthoyl-CoA+H₂O→1,4-dihydroxy-2-naphthoate+CoA

These enzymes are also often referred to as Ydil thioesterases. Enzymesof this family occur in a variety of organisms and have, e.g., beendescribed in Escherichia coli and Salmonella enterica.

Thus, particularly preferred acyl-CoA hydrolases for the enzymaticconversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid of thepresent invention are enzymes which belong to the family of1,4-dihydroxy-2-naphthoyl-CoA hydrolases, more preferably the1,4-dihydroxy-2-naphthoyl-CoA hydrolase derived from Escherichia coli(SEQ ID NO:82) or the 1,4-dihydroxy-2-naphthoyl-CoA hydrolase derivedfrom Salmonella enterica (SEQ ID NO:83).

In a particularly preferred embodiment, the acyl-CoA hydrolase employedin the method of the invention has an amino acid sequence as shown inany one of SEQ ID NOs: 30 to 34 and SEQ ID NOs:82 and 83 or shows anamino acid sequence which is at least x % homologous to any one of SEQID NOs: 30 to 34 and SEQ ID NOs:82 and 83 and has the activity of anacyl-CoA hydrolase with x being an integer between 30 and 100,preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93,94, 95, 96, 97, 98 or 99 wherein such an enzyme is capable of catalyzingthe conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid. Asregards the determination of the sequence identity, the same applies ashas been set forth above.

As described above, the direct conversion of 3-methylcrotonyl-CoA into3-methylcrotonic acid can also be achieved by making use of an enzymewhich is classified as a CoA-transferase (EC 2.8.3.-) capable oftransferring the CoA group of 3-methylcrotonyl-CoA to a carboxylic acid.

CoA-transferases are found in organisms from all lines of descent. Mostof the CoA-transferases belong to two well-known enzyme families(referred to in the following as families I and II) and there exists athird family which had been identified in anaerobic metabolic pathwaysof bacteria. A review describing the different families can be found inHeider (FEBS Letters 509 (2001), 345-349).

Family I contains, e.g., the following CoA-transferases:

For 3-oxo acids: enzymes classified in EC 2.8.3.5 or EC 2.8.3.6;

For short chain fatty acids: enzymes classified in EC 2.8.3.8 or EC2.8.3.9;

For succinate: succinyl-CoA:acetate CoA-transferases, i.e. enzymesclassified in EC 2.8.3.18 (see also Mullins et al., Biochemistry51(2012), 8422-34; Mullins et al., J. Bacteriol. 190 (2006), 4933-4940).

Most enzymes of family I naturally use succinyl-CoA or acetyl-CoA as CoAdonors.

These enzymes contain two dissimilar subunits in different aggregationstates. Two conserved amino acid sequence motives have been identified:

Prosites entry PS01273(http://prosite.expasy.org/cgi-bin/prosite/prosite-search-ac?PDOC00980)

COA_TRANSF_1, PS01273; Coenzyme A transferases signature 1 (PATTERN)

Consensus pattern:

[DN]-[GN]-x(2)-[LIVMFA](3)-G-G-F-x(3)-G-x-P

and

Prosites entries PS01273(http://prosite.expasy.org/cgi-bin/prosite/prosite-search-ac?PDOC00980)

COA_TRANSF_2, PS01274; Coenzyme A transferases signature 2 (PATTERN)

Consensus pattern:

[LF]-[HQ]-S-E-N-G-[LIVF](2)-[GA]

E (glutamic acid) is an active site residue.

The family II of CoA-transferases consists of the homodimeric a-subunitsof citrate lyase (EC 2.8.3.10) and citramalate lyase (EC 2.8.3.11).These enzymes catalyse the transfer of acyl carrier protein (ACP) whichcontains a covalently bound CoA-derivative. It was shown that suchenzymes also accept free CoA-thioester in vitro, such as acetyl-CoA,propionyl-CoA, butyryl-CoA in the case of citrate lyase (Dimroth et al.,Eur. J. Biochem. 80 (1977), 479-488) and acetyl-CoA and succinyl-CoA inthe case of citramalate lyase (Dimroth et al., Eur. J. Biochem. 80(1977), 469-477).

According to Heider (loc. cit.) family III of CoA-transferases consistsof formyl-CoA: oxalate CoA-transferase, succinyl-CoA:(R)-benzylsuccinateCoA-transferase, (E)-cinnamoyl-CoA:(R)-phenyllactate CoA-transferase andbutyrobetainyl-CoA:(R)-carnitine CoA-transferase. A further member offamily III is succinyl-CoA:L-malate CoA-transferase whose function inautrophic CO2 fixation of Chloroflexus aurantiacus is to activateL-malate to its CoA thioester with succinyl-CoA as the CoA donor(Friedman S. et al. J. Bacteriol. 188 (2006), 2646-2655). The amino acidsequences of the CoA-tranferase of this family show only a low degree ofsequence identity to those of families I and II. These CoA-transferasesoccur in prokaryotes and eukaryotes.

In a preferred embodiment the CoA-transferase employed in a methodaccording to the present invention is a CoA-transferase which belongs tofamily I or II as described herein-above.

Preferably, the CoA-transferase employed in a method according to thepresent invention for the direct conversion of 3-methylcrotonyl-CoA into3-methylcrotonic acid is selected from the group consisting of:

-   -   propionate:acetate-CoA transferase (EC 2.8.3.1);    -   acetate CoA-transferase (EC 2.8.3.8); and    -   butyrate-acetoacetate CoA-transferase (EC 2.8.3.9).

Thus, in one preferred embodiment the direct conversion of3-methylcrotonyl-CoA into 3-methylcrotonic acid is achieved by makinguse of an acetate CoA-transferase (EC 2.8.3.8). Acetate CoA-transferasesare enzymes which catalyze the following reaction:

Acyl-CoA+acetate

a fatty acid anion+acetyl-CoA

This enzyme occurs in a variety of bacteria and has, e.g., beendescribed in Anaerostipes caccae, Eubacterium hallii, Faecalibacteriumprausnitzii, Roseburia hominis, Roseburia intestinalis, Coprococcus sp.and Escherichia coli.

In another preferred embodiment the direct conversion of3-methylcrotonyl-CoA into 3-methylcrotonic acid is achieved by makinguse of a butyrate-acetoacetate CoA-transferase (EC 2.8.3.9).Butyrate-acetoacetate CoA-transferase are enzymes which catalyze thefollowing reaction:

Butanoyl-CoA+acetoacetate

butanoate+acetoacetyl-CoA

This enzyme occurs in a variety of organism, including eukaryotic andprokaryotic organisms, such as animals and bacteria. The enzyme has,e.g., been described in Bos taurus, Clostridium sp. and Escherichiacoli.

In another preferred embodiment the direct conversion of3-methylcrotonyl-CoA into 3-methylcrotonic acid is achieved by makinguse of a propionate:acetate-CoA transferase (EC 2.8.3.1).Propionate:acetate-CoA transferases are enzymes which catalyze thefollowing reaction:

Acetyl-CoA+propanoate

acetate+propanoyl-CoA

This enzyme occurs in a variety of organism including prokaryoticorganisms and the enzyme has, e.g., been described in Clostridiumkluyveri, Clostridium propionicum, Clostridium propionicum JCM1430,Cupriavidus necator and Emericella nidulans.

In another preferred embodiment the direct conversion of3-methylcrotonyl-CoA into 3-methylcrotonic acid is achieved by makinguse of a succinyl-CoA:acetate-CoA transferase (EC 2.8.3.18).Succinyl-CoA:acetate CoA-transferases are enzymes which catalyze thefollowing reaction:

Succinyl-CoA+acetate

acetyl-CoA+succinate

This enzyme occurs in a variety of organism, including prokaryoticorganisms, and the enzyme has, e.g., been described in Acetobacteraceti, Trichomonas vaginalis, Tritrichomonas foetus, Tritrichomonasfoetus ATCC 30924 and Trypanosoma brucei.

In another preferred embodiment, the direct conversion of3-methylcrotonyl-CoA into 3-methylcrotonic acid is achieved by makinguse of a CoA-transferase derived from Megasphaera sp. (Uniprot accessionnumber S7HFR5), an enzyme which belongs to the of CoA-transferases (EC2.8.3.-) as defined herein-above.

In a preferred embodiment, the CoA-transferase employed in the method ofthe present invention is a CoA-transferase derived from Megasphaera sp.(Uniprot accession number S7HFR5; SEQ ID NO:84).

In a preferred embodiment of the present invention the CoA-transferaseis an enzyme comprising the amino acid sequence of SEQ ID NO: 84 or asequence which is at least n % identical to SEQ ID NO: 84 with n beingan integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98or 99 and wherein the enzyme has the enzymatic activity of directlyconverting 3-methylcrotonyl-CoA into 3-methylcrotonic acid. As regardsthe determination of the sequence identity, the same applies as has beenset forth above.

The Enzymatic Conversion of 3-Methylcrotonyl-CoA into 3-MethylcrotonicAcid: an Alternative Route to the Above-Described Step VI

In another preferred embodiment, the conversion of 3-methylcrotonyl-CoAinto 3-methylcrotonic acid is achieved by an alternative route wherein3-methylcrotonyl-CoA is first enzymatically converted into3-methylbutyryl-CoA which is then enzymatically converted into3-methylbutyric acid which is then ultimately converted into3-methylcrotonic acid. This alternative conversion of3-methylcrotonyl-CoA into 3-methylcrotonic acid via 3-methylbutyryl-CoAand 3-methylbutyric acid is schematically illustrated in FIG. 32.

Accordingly, the present invention relates to a method for producingisobutene from 3-methylcrotonyl-CoA in which 3-methylcrotonyl-CoA isfirst enzymatically converted into 3-methylbutyryl-CoA which is thenenzymatically converted into 3-methylbutyric acid which is thenconverted into 3-methylcrotonic acid which is then further convertedinto isobutene as described herein above.

The first enzymatic conversion, i.e., the conversion of3-methylcrotonyl-CoA into 3-methylbutyryl-CoA, is a desaturationreaction, i.e., reduction of the double bond C═C of 3-methylcrotonyl-CoAinto 3-methylbutyryl-CoA. The enzymatic conversion of3-methylcrotonyl-CoA into 3-methylbutyryl-CoA, i.e. the reduction of thedouble bond in 3-methylcrotonyl-CoA, can, for example, be achieved byemploying an enzyme classified as EC 1.3._._. Enzymes classified as EC1.3._._ are oxidoreductases acting on the CH—CH group of a donormolecule. This subclass contains enzymes that reversibly catalyze theconversion of a carbon-carbon single bond to a carbon-carbon double bondbetween two carbon atoms. Sub-classes of EC 1.3 are classified dependingon the acceptor. In one preferred embodiment the enzyme is an enzymewhich is classified as EC 1.3._._ and which uses NADH or NADPH asco-factor. In one particularly preferred embodiment the enzyme is anenzyme which uses NADPH as a co-factor. In a preferred embodiment theenzyme is selected from the group consisting of:

-   -   acyl-CoA dehydrogenase (NADP+) (EC 1.3.1.8);    -   enoyl-[acyl-carrier-protein] reductase (NADPH, Si-specific) (EC        1.3.1.10);    -   cis-2-enoyl-CoA reductase (NADPH) (EC 1.3.1.37);    -   trans-2-enoyl-CoA reductase (NADPH) (EC 1.3.1.38);    -   enoyl-[acyl-carrier-protein] reductase (NADPH, Re-specific) (EC        1.3.1.39); and    -   crotonyl-CoA reductase (EC 1.3.1.86).

Thus, in one preferred embodiment the conversion of 3-methylcrotonyl-CoAinto 3-methylbutyryl-CoA is achieved by making use of an acyl-CoAdehydrogenase (NADP+) (EC 1.3.1.8). Acyl-CoA dehydrogenases are enzymeswhich catalyze the following reaction:

Acyl-CoA+NADP⁺

2,3-dehydroacyl-CoA+NADPH+H⁺

This enzyme occurs in a variety of organism, including eukaryotic andprokaryotic organisms, such as plants, animals, fungi and bacteria. Theenzyme has, e.g., been described in Bos, taurus, Rattus novegicus, Musmusculus, Columba sp., Arabidopsis thaliana, Nicotiana benthamiana,Allium ampeloprasum, Euglena gracilis, Candida albicans, Streptococcuscollinus, Rhodobacter sphaeroides and Mycobacterium smegmatis.

In a further preferred embodiment the conversion of 3-methylcrotonyl-CoAinto 3-methylbutyryl-CoA is achieved by making use of anenoyl-[acyl-carrier-protein] reductase (NADPH, Si-specific) (EC1.3.1.10). Enoyl-[acyl-carrier-protein] reductases (NADPH, Si-specific)are enzymes which catalyze the following reaction:

acyl-[acyl-carrier-protein]+NADP⁺

trans-2,3-dehydroacyl-[acyl-carrier-protein]+NADPH+H⁺

This enzyme occurs in a variety of organism, including eukaryotic andprokaryotic organisms, such as plants, fungi and bacteria. The enzymehas, e.g., been described in Carthamus tinctorius, Candida tropicalis,Saccharomyces cerevisiae, Streptococcus collinus, Streptococcuspneumoniae, Staphylococcus aureus, Bacillus subtilis, Bacillus cereus,Porphyromonas gingivalis, Escherichia coli and Salmonella enterica.

In a further preferred embodiment the conversion of 3-methylcrotonyl-CoAinto 3-methylbutyryl-CoA is achieved by making use of a cis-2-enoyl-CoAreductase (NADPH) (EC 1.3.1.37). Cis-2-enoyl-CoA reductases (NADPH) areenzymes which catalyze the following reaction:

Acyl-CoA+NADP⁺

cis-2,3-dehydroacyl-CoA+NADPH+H⁺

This enzyme has been described to occur in Escherichia coli.

In a further preferred embodiment the conversion of 3-methylcrotonyl-CoAinto 3-methylbutryryl-CoA is achieved by making use of atrans-2-enoyl-CoA reductase (NADPH) (EC 1.3.1.38). Trans-2-enoyl-CoAreductases (NADPH) are enzymes which catalyze the following reaction:

Acyl-CoA+NADP⁺

trans-2,3-dehydroacyl-CoA+NADPH+H⁺

This enzyme occurs in a variety of organism, including eukaryotic andprokaryotic organisms, such as plants, animals and bacteria. The enzymehas, e.g., been described in Homo sapien, Rattus norvegicus, Musmusculus, Cavia porcellus, Caenorhabditis elegans, Phalaenopsisamabilis, Gossypium hirsutum, Mycobacterium tuberculosis, Streptococcuscollinu and Escherichia coli.

In a further preferred embodiment the conversion of 3-methylcrotonyl-CoAinto 3-methylbutyryl-CoA is achieved by making use of anenoyl-[acyl-carrier-protein] reductase (NADPH, Re-specific) (EC1.3.1.39). Enoyl-[acyl-carrier-protein] reductases (NADPH, Re-specific)are enzymes which catalyze the following reaction:acyl-[acyl-carrier-protein]+NADP⁺

trans-2,3-dehydroacyl-[acyl-carrier-protein]+NADPH+H⁺

This enzyme occurs in a variety of organism, including eukaryotic andprokaryotic organisms, such as animals and bacteria. The enzyme has,e.g., been described in Gallus gallus, Pigeon, Rattus norvegicus, Caviaporcellus, Staphylococcus aureus, Bacillus subtilis and Porphyromonasgingivalis.

In a further preferred embodiment the conversion of 3-methylcrotonyl-CoAinto 3-methylbutyryl-CoA is achieved by making use of a crotonyl-CoAreductase (EC 1.3.1.86). Crotonyl-CoA reductases are enzymes whichcatalyze the following reaction:

butanoyl-CoA+NADP⁺

(E)-but-2-enoyl-CoA+NADPH+H⁺

This enzyme occurs in a variety of organism, including eukaryotic andprokaryotic organisms, such as animals, fungi and bacteria. The enzymehas, e.g., been described in Bos taurus, Salinospora tropica,Clostridium difficile, Streptomyces collinus, Streptomyces cinnamonensisand Streptomyces hygroscopicus.

The second enzymatic conversion, i.e., the conversion of3-methylbutyryl-CoA into 3-methylbutyric acid, can be achieved bydifferent enzymatic conversions. One possibility is the directconversion via a hydrolysis reaction. Another possibility is the directconversion via a reaction catalyzed by a CoA-transferase and a thirdpossibility is a two-step conversion via 3-methylbutyryl phosphate.

Thus, according to the present invention, the enzymatic conversion of3-methylbutyryl-CoA into 3-methylbutyric acid is achieved by

-   -   (a) a single enzymatic reaction in which 3-methylbutyryl-CoA is        directly converted into 3-methylbutyric acid, preferably by        making use of a CoA transferase (EC 2.8.3.-), preferably a        propionate:acetate-CoA transferase (EC 2.8.3.1), an acetate        CoA-transferase (EC 2.8.3.8) or a succinyl-CoA:acetate        CoA-transferase (EC 2.8.3.18);    -   (b) a single enzymatic reaction in which 3-methylbutyryl-CoA is        directly converted into 3-methylbutyric acid, preferably by        making use of a thioester hydrolase (EC 3.1.2.-), preferably        acetyl-CoA hydrolase (EC 3.1.2.1), an ADP-dependent        short-chain-acyl-CoA hydrolase (EC 3.1.2.18) or an acyl-CoA        hydrolase (EC 3.1.2.20); or    -   (c) two enzymatic steps comprising        -   (i) first enzymatically converting 3-methylbutyryl-CoA into            3-methylbutyryl phosphate; and        -   (ii) then enzymatically converting the thus obtained            3-methylbutyryl phosphate into said 3-methylbutyric acid.

As regards the preferred embodiments for the CoA transferase (EC2.8.3.-), the propionate:acetate-CoA transferase (EC 2.8.3.1), theacetate CoA-transferase (EC 2.8.3.8) or a succinyl-CoA:acetateCoA-transferase (EC 2.8.3.18), the thioester hydrolase (EC 3.1.2.-), theacetyl-CoA hydrolase (EC 3.1.2.1), the ADP-dependentshort-chain-acyl-CoA hydrolase (EC 3.1.2.18), the acyl-CoA hydrolase (EC3.1.2.20), the enzyme capable of converting 3-methylbutyryl-CoA into3-methylbutyryl phosphate and the enzyme capable of converting3-methylbutyryl phosphate into said 3-methylbutyric acid, the sameapplies as has been set forth above in connection with the enzymaticconversion of step VIa, step VIb and step VIc according to theinvention.

The third enzymatic conversion, i.e., the conversion of 3-methylbutyricacid into 3-methylcrotonic acid can, e.g., be achieved by a 2-enoatereductase (EC 1.3.1.31). 2-enoate reductases are enzymes which naturallycatalyze the following reaction:

Butanoate+NAD⁺

^(but-)2-enoate+NADH+H⁺

This enzyme occurs in a variety of organism, including eukaryotic andprokaryotic organisms, such as animals, fungi and bacteria. The enzymehas, e.g., been described in Cichorium intybus, Marchantia polymorpha,Solanum lycopersicum, Absidia glauca, Kluyveromyces lactis, Penicilliumcitrinum; Rhodosporidium, Saccharomyces cerevisiae, Clostridiumkluyveri, Clostridium bifermentans, Clostridium botulinum, Clostridiumdifficile, Clostridium ghonii, Clostridium mangenotii, Clostridiumoceanicum, Clostridium sordellii, Clostridium sporogenes, Clostridiumsticklandii, Clostridium tyrobutyricum, Achromobacter sp., Burkholderiasp., Gluconobacter oxydans, Lactobacillus casei, Pseudomonas putida,Shewanella sp., Yersinia bercovieri, Bacillus subtilis, Moorellathermoacetica and Peptostreptococcus anaerobius. The enoate reductase ofClostridiae has been described, e.g., in Tischler et al. (Eur. J.Bioche. 97 (1979), 103-112).

The Enzymatic Conversion of 3-Methylglutaconyl-CoA into3-Methylcrotonyl-CoA: Step VII as Shown in FIG. 1

The 3-methylcrotonyl-CoA which is converted according to the method ofthe present invention into 3-methylcrotonic acid according to any of theabove described methods (and further converted according to the methodof the present invention into isobutene according to any of the abovedescribed methods) may itself be provided by an enzymatic reaction,namely the enzymatic conversion of 3-methylglutaconyl-CoA into3-methylcrotonyl-CoA. The conversion of 3-methylglutaconyl-CoA into3-methylcrotonyl-CoA is schematically illustrated in FIG. 12.

Accordingly, the present invention relates to a method for producingisobutene from 3-methylglutaconyl-CoA in which 3-methylglutaconyl-CoA isfirst converted into 3-methylcrotonyl-CoA which is then furtherconverted into 3-methylcrotonic acid which is then further convertedinto isobutene as described herein above.

The conversion of 3-methylglutaconyl-CoA into 3-methylcrotonyl-CoA maybe catalyzed by different enzymes. According to the present invention,the conversion of 3-methylglutaconyl-CoA into said 3-methylcrotonyl-CoApreferably makes use of (i) a methylcrotonyl-CoA carboxylase (EC6.4.1.4); or (ii) a geranoyl-CoA carboxylase (EC 6.4.1.5) (as shown instep VII of FIG. 1).

Methylcrotonyl-CoA carboxylases (EC 6.4.1.4) and geranoyl-CoAcarboxylases (EC 6.4.1.5) as well as preferred enzymes of these enzymeclasses have already been described above. Accordingly, as regards theseenzymes, the same applies to the conversion of 3-methylglutaconyl-CoAinto 3-methylcrotonyl-CoA as has been set forth above.

In another preferred embodiment the conversion of 3-methylglutaconyl-CoAvia decarboxylation into 3-methylcrotonyl-CoA is catalyzed by a3-methylglutaconyl-CoA decarboxylase, e.g. a 3-methylglutaconyl-CoAdecarboxylase of Myxococcus xanthus encoded by the liuB gene. This genecodes for an enzyme having the two subunits AibA and AibB (Li et al.,Angew. Chem. Int. Ed. 52 (2013), 1304-1308).

This enzyme has already described above as a methylcrotonyl-CoAcarboxylase derived from Myxcoxoccus xanthus in the context ofconversion of 3-methylcrotonic acid into isobutene.

The same enzyme derived from Myxococcus xanthus encoded by the liuB genehaving the two subunits AibA and AibB (Li et al., Angew. Chem. Int. Ed.52 (2013), 1304-1308) has been described above with reference to SEQ IDNOs: 100 and 101 and can also be used for the conversion of3-methylglutaconyl-CoA via decarboxylation into 3-methylcrotonyl-CoA.

In a preferred embodiment of the present invention the3-methylglutaconyl-CoA decarboxylase is an enzyme comprising the aminoacid sequence of SEQ ID NO: 100 or a sequence which is at least n %identical to SEQ ID NO: 100 with n being an integer between 10 and 100,preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 and wherein the enzyme hasthe enzymatic activity of converting 3-methylglutaconyl-CoA into3-methylcrotonyl-CoA. As regards the determination of the sequenceidentity, the same applies as has been set forth above.ln anotherpreferred embodiment of the present invention the 3-methylglutaconyl-CoAdecarboxylase is an enzyme comprising the amino acid sequence of SEQ IDNO: 101 or a sequence which is at least n % identical to SEQ ID NO: 101with n being an integer between 10 and 100, preferably 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95,96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity ofconverting 3-methylglutaconyl-CoA into 3-methylcrotonyl-CoA. As regardsthe determination of the sequence identity, the same applies as has beenset forth above.

In another preferred embodiment of the present invention the3-methylglutaconyl-CoA decarboxylase is a heterodimeric enzymecomprising a combination of the amino acid sequence of SEQ ID NO: 100and 101 or a sequence which is at least n identical to SEQ ID NO: 100and 101 with n being an integer between 10 and 100, preferably 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93,94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymaticactivity of converting 3-methylglutaconyl-CoA into 3-methylcrotonyl-CoA.As regards the determination of the sequence identity, the same appliesas has been set forth above.

The enzymatic conversion of 3-hydroxy-3-methylcilutaryl-CoA into3-methylglutaconyl-CoA: step VIII as shown in FIG. 1

The 3-methylglutaconyl-CoA which is converted into 3-methylcrotonyl-CoAmay itself be provided by an enzymatic reaction, namely the enzymaticconversion of 3-hydroxy-3-methylglutaryl-CoA into3-methylglutaconyl-CoA; see FIG. 13.

Accordingly, the present invention also relates to a method forproducing isobutene from 3-hydroxy-3-methylglutaryl-CoA in which3-hydroxy-3-methylglutaryl-CoA is first converted into3-methylglutaconyl-CoA which is then converted into 3-methylcrotonyl-CoAwhich is then further converted into 3-methylcrotonic acid which is thenfurther converted into isobutene as described herein above.

According to the present invention, the enzymatic conversion of3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA is anenzymatic dehydration reaction which occurs naturally, and which iscatalyzed, e.g., by enzymes classified as 3-methylglutaconyl-coenzyme Ahydratase (EC 4.2.1.18). Accordingly, the enzymatic conversion of3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA preferablymakes use of a 3-methylglutaconyl-coenzyme A hydratase (EC 4.2.1.18) (asshown in step VIII of FIG. 1).

3-methylglutaconyl-coenzyme A hydratases are enzymes which catalyze thefollowing reaction:

(S)-3-hydroxy-3-methylglutaryl-CoA

trans-3-methylglutaconyl-CoA+H₂O

This enzyme occurs in a variety of organisms, including eukaryotic andprokaryotic organisms, such as plants, animals and bacteria. The enzymehas, e.g., been described in Catharantus roseus, Homo sapiens, Bostaurus, Ovis aries, Acinetobacter sp., Myxococcus sp. and Pseudomonasputida. In a preferred embodiment the 3-methylglutaconyl-coenzyme Ahydratase is an enzyme from Myxococcus sp., and even more preferably anenzyme which has an amino acid sequence as shown in SEQ ID NO: 35 orshows an amino acid sequence which is at least x % homologous to SEQ IDNO: 35 and has the activity of a 3-methylglutaconyl-coenzyme A hydratasewith x being an integer between 30 and 100, preferably 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99wherein such an enzyme is capable of converting3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA as set forthherein above. As regards the determination of the degree of identity,the same applies as has been set forth herein above.

The conversion of 3-hydroxy-3-methylglutaryl-CoA into3-methylglutaconyl-CoA can also be achieved by making use of a3-hydroxy-3-methylglutaryl-coenzyme A dehydratase activity which hasbeen identified, e.g., in Myxococcus xanthus and which is encoded by theliuC gene (Li et al., Angew. Chem. Int. Ed. 52 (2013), 1304-1308). The3-hydroxy-3-methylglutaryl-coenzyme A dehydratase derived fromMyxococcus xanthus has the Uniprot Accession number Q1 D5Y4. Thus, in apreferred embodiment, the 3-hydroxy-3-methylglutaryl-coenzyme Adehydratase employed in the method of the present invention is an enzymederived from Myxococcus xanthus (Uniprot Accession number Q1 D5Y4; SEQID NO:98).

In a preferred embodiment of the present invention the3-hydroxy-3-methylglutaryl-coenzyme A dehydratase is an enzymecomprising an amino acid sequence of SEQ ID NO:98 or a sequence which isat least n % identical to SEQ ID NO:98 with n being an integer between10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 and wherein theenzyme has the enzymatic activity of converting3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA. As regardsthe determination of the sequence identity, the same applies as has beenset forth above.

The enzymatic conversion of 3-hydroxy-3-methylglutaryl-CoA into3-methylglutaconyl-CoA can also be achieved by making use of a3-hydroxyacyl-CoA dehydratase or an enoyl-CoA hydratase.3-hydroxyacyl-CoA dehydratases and enoyl-CoA hydratases catalyze thesame reaction while the name of one of these enzymes denotes onedirection of the corresponding reaction while the other name denotes thereverse reaction. As the reaction is reversible, both enzyme names canbe used.

3-hydroxyacyl-CoA dehydratases and enoyl-CoA hydratases belong toenzymes classified as EC 4.2.1.-.

3-hydroxyacyl-CoA dehydratases and enoyl-CoA hydratases have, e.g., beenidentified in Pseudomonas sp., Acinetobacter baumanii (Uniprot accessionnumber A0A0D5YDD4), Pseudomonas aeruginosa (Uniprot accession numberQ9HZV7), Marinobacter santoriniensis (Uniprot accession number M7CV63),Pseudomonas knackmussii, Pseudomonas pseudoalcaligenes (Uniprotaccession number L8MQT6), Pseudomonas flexibilis and Alcanivoraxdieselolei as well as in Ustilago maydis (Uniprot accession numberQ4PEN0), Bacillus sp. GeD10 (Uniprot accession number N1LWG2) and inLabilithrix luteola (Uniprot accession number A0A0K1PN19).

In a preferred embodiment, the 3-hydroxyacyl-CoA dehydratase/enoyl-CoAhydratase employed in the method of the present invention for theconversion of 3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoAis an enzyme derived from Pseudomonas sp. (SEQ ID NO:85), Acinetobacterbaumanii (Uniprot accession number A0A0D5YDD4; SEQ ID NO:86),Pseudomonas aeruginosa (Uniprot accession number Q9HZV7; SEQ ID NO:87),Marinobacter santoriniensis (Uniprot accession number Q9HZV7; SEQ IDNO:88), Pseudomonas knackmussii (SEQ ID NO:89), Pseudomonaspseudoalcaligenes (Uniprot accession number L8MQT6; SEQ ID NO:90),Pseudomonas flexibilis (SEQ ID NO:91), Alcanivorax dieselolei (SEQ IDNO:92), Ustilago maydis (Uniprot accession number Q4PENO; SEQ ID NO:95),Bacillus sp. GeD10 (Uniprot accession number N1LWG2; SEQ ID NO:96) orLabilithrix luteola (Uniprot accession number A0A0K1PN19; SEQ ID NO:97).

In a preferred embodiment of the present invention the 3-hydroxyacyl-CoAdehydratase/enoyl-CoA hydratase is an enzyme comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 85 to 92 andSEQ ID NOs: 95 to 97 or a sequence which is at least n % identical toany of SEQ ID NOs: 85 to 92 and SEQ ID NOs: 95 to 97 with n being aninteger between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99and wherein the enzyme has the enzymatic activity of converting3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA. As regardsthe determination of the sequence identity, the same applies as has beenset forth above.

The Enzymatic Conversion of Acetoacetyl-CoA into3-Hydroxy-3-Methylglutaryl-CoA: Step IX as Shown in FIG. 1

The 3-hydroxy-3-methylglutaryl-CoA which is converted into3-methylglutaconyl-CoA may itself be provided by an enzymatic reaction,namely the enzymatic condensation of acetoacetyl-CoA and acetyl-CoA into3-hydroxy-3-methylglutaryl-CoA; see FIG. 14.

Accordingly, the present invention also relates to a method forproducing isobutene from acetoacetyl-CoA and acetyl-CoA in whichacetoacetyl-CoA and acetyl-CoA are first condensed into3-hydroxy-3-methylglutaryl-CoA which is then converted into3-methylglutaconyl-CoA which is then converted into 3-methylcrotonyl-CoAwhich is then further converted into 3-methylcrotonic acid which is thenfurther converted into isobutene as described herein above.

According to the present invention, the enzymatic condensation ofacetoacetyl-CoA and acetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA makespreferably use of a 3-hydroxy-3-methylglutaryl-CoA synthase (see step IXof FIG. 1).

The condensation of acetyl-CoA and acetoacetyl-CoA is a reaction whichis naturally catalyzed by the enzyme 3-hydroxy-3-methylglutaryl-CoAsynthase (also referred to as HMG-CoA synthase). Thus, preferably, thecondensation of acetyl-CoA and acetoacetyl-CoA into3-hydroxy-3-methylglutaryl-CoA makes use of a3-hydroxy-3-methylglutaryl-CoA synthase (also referred to as HMG-CoAsynthase). HMG-CoA synthases are classified in EC 2.3.3.10 (formerly,HMG-CoA synthase has been classified as EC 4.1.3.5 but has beentransferred to EC 2.3.3.10). The term “HMG-CoA synthase” refers to anyenzyme which is able to catalyze the reaction where acetyl-CoA condenseswith acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA)(see FIG. 14). HMG-CoA synthase is part of the mevalonate pathway. Twopathways have been identified for the synthesis of isopentenylpyrophosphate (IPP), i.e. the mevalonate pathway and the glyceraldehyde3-phosphate-pyruvate pathway. HMG-CoA synthase catalyzes the biologicalClaisen condensation of acetyl-CoA with acetoacetyl-CoA and is a memberof a superfamily of acyl-condensing enzymes that includesbeta-ketothiolases, fatty acid synthases (beta-ketoacyl carrier proteinsynthase) and polyketide synthases.

HMG-CoA synthase has been described for various organisms. Also aminoacid and nucleic acid sequences encoding HMG-CoA synthases from numeroussources are available. Generally, the sequences only share a low degreeof overall sequence identity. For example, the enzymes fromStaphylococcus or Streptococcus show only about 20% identity to those ofhuman and avian HMG-CoA synthase. In some sources it is reported thatthe bacterial HMG-CoA synthases and their animal counterparts exhibitonly about 10% overall sequence identity (Sutherlin et al., J.Bacteriol. 184 (2002), 4065-4070). However, the amino acid residuesinvolved in the acetylation and condensation reactions are conservedamong bacterial and eukaryotic HMG-CoA synthases (Campobasso et al., J.Biol. Chem. 279 (2004), 44883-44888). The three-dimensional structure ofthree HMG-CoA synthase enzymes has been determined and the amino acidscrucial for the enzymatic reaction are in principle well characterized(Campobasso et al., loc. cit.; Chun et al., J. Biol. Chem. 275 (2000),17946-17953; Nagegowda et al., Biochem. J. 383 (2004), 517-527; Hegardt,Biochem. J. 338 (1999), 569-582). In eukaryotes, there exist two formsof the HMG-CoA synthase, i.e. a cytosolic and a mitochondrial form. Thecytosolic form plays a key role in the production of cholesterol andother isoprenoids and the mitochondrial form is involved in theproduction of ketone bodies.

In principle any HMG-CoA synthase enzyme can be used in the context ofthe present invention, in particular from prokaryotic or eukaryoticorganisms.

Prokaryotic HMG-CoA synthases are described, e.g., from Staphylococcusaureus (Campobasso et al., loc. cit.; Uniprot accession number Q9FD87),Staphylococcus epidermidis (Uniprot accession number Q9FD76),Staphylococcus haemolyticus (Uniprot accession number Q9FD82),Enterococcus faecalis (Sutherlin et al., loc. cit.; Unirprot accessionnumber Q9FD71; SEQ ID NO:99), Enterococcus faecium (Uniprot accessionnumber Q9FD66), Streptococcus pneumonia (Uniprot accession numberQ9FD56), Streptococcus pyogenes (Uniprot accession number Q9FD61) andMethanobacterium thermoautotrophicum (accession number AE000857),Borrelia burgdorferi (NCBI accession number BB0683). Further HMG-CoAsynthases are, e.g., described in WO 2011/032934. A preferred HMG-CoAsynthase is the enzyme from Schizosaccharomyces pombe (Uniprot P54874).In a particularly preferred embodiment, the HMG-CoA synthase employed inthe method of the invention has an amino acid sequence as shown in SEQID NO: 36 or SEQ ID NO:99 or shows an amino acid sequence which is atleast x % homologous to SEQ ID NO: 36 or SEQ ID NO:99 and has theactivity of a HMG-CoA synthase with x being an integer between 30 and100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92,93, 94, 95, 96, 97, 98 or 99 wherein such an enzyme is capable ofcatalyzing the condensation of acetyl-CoA and acetoacetyl-CoA into3-hydroxy-3-methylglutaryl-CoA. As regards the determination of thedegree of identity, the same applies as has been set forth herein above.

The Enzymatic Conversion of Acetyl-CoA into Acetoacetyl-CoA: Steps XIII,XIV and XV as Shown in FIG. 1

The acetoacetyl-CoA which is either converted into3-hydroxy-3-methylglutaryl-CoA or which is converted into acetoacetatemay itself be provided by an enzymatic reaction, namely the enzymaticconversion of acetyl-CoA into acetoacetyl-CoA.

According to the present invention, the conversion of acetyl-CoA intosaid acetoacetyl-CoA can be achieved by different routes. Onepossibility is to first convert acetyl-CoA into malonyl-CoA (step XIV asshown in FIG. 1) and then to further condense said malonyl-CoA andacetyl-CoA into acetoacetyl-CoA (step XV as shown in FIG. 1). Anotherpossibility is to directly condense in a single enzymatic reaction twomolecules of acetyl-CoA into acetoacetyl-CoA (step XIII as shown in FIG.1). These reactions are schematically shown in FIG. 15 (step XIII), FIG.16 (step XIV) and FIG. 17 (step XV), respectively.

Thus, the present invention also relates to a method for producingisobutene from acetyl-CoA in which acetyl-CoA is first converted intoacetoacetyl-CoA by any of the above-mentioned routes which is thencondensed with acetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA which isthen converted into 3-methylglutaconyl-CoA which is then converted into3-methylcrotonyl-CoA which is then further converted into3-methylcrotonic acid which is then further converted into isobutene asdescribed herein above.

Moreover, the present invention also relates to a method for producingisobutene from acetyl-CoA in which acetyl-CoA is first converted intoacetoacetyl-CoA by any of the above-mentioned routes by any of theabove-mentioned routes which is then converted into acetoacetate whichis then converted into acetone which is then condensed with acetyl-CoAinto 3-hydroxyisovalerate (HIV) which is then converted into3-methylcrotonic acid as described herein above. Further, said3-methylcrotonic acid is then further converted into isobutene asdescribed herein above.

According to the present invention, the enzymatic conversion ofacetyl-CoA into malonyl-CoA preferably makes use of an acetyl-CoAcarboxylase (EC 6.4.1.2) (step XIV as shown in FIG. 1). This naturallyoccurring reaction fixes CO₂ on acetyl-CoA utilizing ATP resulting inmalonyl-CoA. Enzymes classified as an acetyl-CoA carboxylases (EC6.4.1.2) catalyze the following reaction:

Acetyl-CoA+ATP+CO₂→Malonyl-CoA+ADP

Moreover, according to the present invention, the enzymatic condensationof malonyl-CoA and acetyl-CoA into said acetoacetyl-CoA preferably makesuse of an acetoacetyl-CoA synthase (EC 2.3.1.194) (step XV as shown inFIG. 1). This is a natural occurring reaction and condenses malonyl-CoAand acetyl-CoA in a decarboxylation reaction. Enzymes classified asacetoacetyl-CoA synthases (EC 2.3.1.194) catalyze the enzymaticconversion of acetyl-CoA and malonyl-CoA into acetoacetyl-CoA accordingto the following reaction.

acetyl-CoA+malonyl-CoA→acetoacetyl-CoA+CoA+CO₂

This reaction is catalyzed by an enzyme called acetoacetyl-CoA synthase(EC 2.3.1.194). The gene encoding this enzyme was identified in themevalonate pathway gene cluster for terpenoid production in asoil-isolated Gram-positive Streptomyces sp. Strain CL190 (Okamura etal., PNAS USA 107 (2010), 11265-11270, 2010). Moreover a biosyntheticpathway using this enzyme for acetoacetyl-CoA production was recentlydeveloped in E. coli (Matsumoto K et al., Biosci. Biotechnol. Biochem,75 (2011), 364-366).

Alternatively, the enzymatic conversion of acetyl-CoA into saidacetoacetyl-CoA consists of a single enzymatic reaction in whichacetyl-CoA is directly converted into acetoacetyl-CoA by the enzymaticcondensation of two molecules of acetyl-CoA into acetoacetyl-CoA.Preferably, the enzymatic conversion of acetyl-CoA into acetoacetyl-CoAis achieved by making use of an acetyl-CoA acetyltransferase (EC2.3.1.9).

Thus, acetoacetyl-CoA can be produced from acetyl-CoA as, e.g.,described in WO 2013/057194. Therefore, according to the presentinvention, acetyl-CoA can, for example, be converted intoacetoacetyl-CoA by the following reaction:

2 acetyl-CoA

acetoacetyl-CoA+CoA

This reaction is a naturally occurring reaction and is catalyzed byenzymes called acetyl-CoA C-acetyltransferases which are classified asEC 2.3.1.9. Enzymes belonging to this class and catalyzing the aboveshown conversion of two molecules of acetyl-CoA into acetoacetyl-CoA andCoA occur in organisms of all kingdoms, i.e. plants, animals, fungi,bacteria etc. and have extensively been described in the literature.Nucleotide and/or amino acid sequences for such enzymes have beendetermined for a variety of organisms, like Homo sapiens, Arabidopsisthaliana, E. coli, Bacillus subtilis, Clostridium acetobutylicum andCandida, to name just some examples. In principle, any acetyl-CoAC-acetyltransferase (EC 2.3.1.9) can be used in the context of thepresent invention. In one preferred embodiment the enzyme is anacetyl-CoA acetyltransferase from Clostridium acetobutylicum (UniprotP45359). In a particularly preferred embodiment, the acetyl-CoAacetyltransferase employed in the method of the invention has an aminoacid sequence as shown in SEQ ID NO: 37 or shows an amino acid sequencewhich is at least x % homologous to SEQ ID NO: 37 and has the activityof an acetyl-CoA acetyltransferase with x being an integer between 30and 100, preferably 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91,92, 93, 94, 95, 96, 97, 98 or 99 wherein such an enzyme is capable ofconverting acetyl-CoA into acetoacetyl-CoA as set forth herein above.

As regards the determination of the degree of identity, the same appliesas has been set forth herein above.

The Enzymatic Recycling of Metabolites Occurring in the Pathway of thePresent Invention: Steps Xa, Xb, XI and XII as Shown in FIG. 1

The above-described method of the present invention for producingisobutene from acetyl-CoA may be supplemented by one or more of thefollowing reactions as shown in step Xa, step Xb, step XI and step XIIof FIG. 18.

These steps relate to alternative bioconversions which may occurconcomitantly to any of the above-described methods for producingisobutene.

Thus, the present invention relates to any of the above-describedmethods for producing isobutene from 3-methylcrotonic acid (or from anyof the above-described intermediates in the described pathways fromacetyl-CoA into isobutene) wherein additionally

-   -   a) 3-hydroxyisovalerate (HIV) is enzymatically converted into        3-methylcrotonic acid with a concomitant transfer of CoA from        3-methylcrotonyl-CoA on 3-hydroxyisovalerate (HIV) to result in        3-hydroxyisovaleryl-CoA (step Xa as schematically shown in FIG.        19); and/or    -   b) 3-hydroxyisovalerate (HIV) is enzymatically converted into        3-hydroxyisovaleryl-CoA (step Xb as schematically shown in FIG.        20); and/or    -   c) 3-hydroxyisovaleryl-CoA is enzymatically converted into        3-methylcrotonyl-CoA (step XI as schematically shown in FIG.        21); and/or    -   d) 3-hydroxyisovalerate (HIV) is enzymatically converted into        3-hydroxyisovaleryl-CoA (step XII as schematically shown in FIG.        22).

These reactions which which will be described in more detail in thefollowing, may occur concomitantly to any of the above-described methodsfor producing isobutene are beneficial for several reasons. First, it isknown that the hydration of an enoyl-CoA (such as, e.g.,3-methylcrotonyl-CoA) is a favoured reaction in vivo in an aqueousmedium. Accordingly, the above reactions represent possibilities whichallow to drive the metabolic flux toward the precursor of isobutene,i.e., 3-methylcrotonic acid, even in case the pathway “leaks” into thedirection of 3-hydroxyisovalerate (HIV) and/or 3-hydroxyisvaleryl-CoA.Second, the above conversions beneficially involve the conservation ofenergy into a thioester CoA bond via a transfer of a thioester group.

The enzymatic conversion of 3-hydroxyisovalerate (HIV) into3-methylcrotonic acid with a concomitant transfer of CoA from3-methylcrotonyl-CoA on 3-hydroxyisovalerate (HIV) to result in3-hydroxyisovaleryl-CoA as shown in step Xa of FIG. 18

Thus, in a first aspect, the 3-methylcrotonic acid which is convertedinto isobutene may be provided by an enzymatic reaction wherein3-hydroxyisovalerate (HIV) is enzymatically converted into3-methylcrotonic acid with a concomitant transfer of CoA from3-methylcrotonyl-CoA to 3-hydroxyisovalerate (HIV) to result in3-hydroxyisovaleryl-CoA (step Xa as shown in FIG. 18). This reaction isschematically illustrated in FIG. 19.

Thus, the present invention also relates to a method for producingisobutene from 3-hydroxyisovalerate (HIV) wherein 3-hydroxyisovalerate(HIV) is enzymatically converted into 3-methylcrotonic acid with aconcomitant transfer of CoA from 3-methylcrotonyl-CoA on3-hydroxyisovalerate (HIV) to result in 3-hydroxyisovaleryl-CoA.Further, the thus produced 3-methylcrotonic acid is then enzymaticallyconverted into isobutene as described herein above.

Moreover, the present invention also relates to a method for producing3-methylcrotonic acid and 3-hydroxyisovaleryl-CoA from3-hydroxyisovalerate (HIV) and from 3-methylcrotonyl-CoA wherein3-hydroxyisovalerate (HIV) is enzymatically converted into3-methylcrotonic acid with a concomitant transfer of CoA from3-methylcrotonyl-CoA on 3-hydroxyisovalerate (HIV) to result in3-hydroxyisovaleryl-CoA.

According to the present invention, the conversion of3-hydroxyisovalerate (HIV) and 3-methylcrotonyl-CoA into3-methylcrotonic acid and 3-hydroxyisovaleryl-CoA wherein3-hydroxyisovalerate (HIV) is enzymatically converted into3-methylcrotonic acid with a concomitant transfer of CoA from3-methylcrotonyl-CoA on 3-hydroxyisovalerate (HIV) to result in3-hydroxyisovaleryl-CoA preferably makes use of an enzyme which isclassified as a CoA-transferase (EC 2.8.3.-) capable of transferring theCoA group of 3-methylcrotonyl-CoA to a carboxylic acid, i.e.,3-hydroxyisovalerate (HIV).

CoA-transferases (EC 2.8.3.-) as well as preferred enzymes of thisenzyme class have already been described above. Accordingly, as regardsthese enzymes, the same applies to the conversion of3-hydroxyisovalerate (HIV) and 3-methylcrotonyl-CoA into3-methylcrotonic acid and 3-hydroxyisovaleryl-CoA as has been set forthabove.

Preferably, the CoA-transferase employed in a method according to thepresent invention in the enzymatic conversion of 3-hydroxyisovalerate(HIV) into 3-methylcrotonic acid with a concomitant transfer of CoA from3-methylcrotonyl-CoA on 3-hydroxyisovalerate (HIV) to result in3-hydroxyisovaleryl-CoA is a CoA-transferase selected from the groupconsisting of:

-   -   propionate:acetate-CoA transferase (EC 2.8.3.1);    -   acetate CoA-transferase (EC 2.8.3.8); and    -   butyrate-acetoacetate CoA-transferase (EC 2.8.3.9).

Propionate:acetate-CoA transferases (EC 2.8.3.1), acetateCoA-transferases (EC 2.8.3.8) and butyrate-acetoacetate CoA-transferases(EC 2.8.3.9) as well as preferred enzymes of these enzyme classes havealready been described above. Accordingly, as regards these enzymes, thesame applies to the conversion of 3-hydroxyisovalerate (HIV) and3-methylcrotonyl-CoA into 3-methylcrotonic acid and3-hydroxyisovaleryl-CoA as has been set forth above.

The Enzymatic Conversion of 3-Hydroxyisovalerate (HIV) into3-Hydroxyisovaleryl-CoA as Shown in step Xb of FIG. 18

In addition or in the alternative to the above-described methods (stepXa), the 3-hydroxyisovaleryl-CoA may also be provided by an enzymaticconversion of 3-hydroxyisovalerate into said 3-hydroxyisovaleryl-CoA(step Xb as shown in FIG. 18). In this reaction, 3-hydroxyisovaleratereacts with an acyl-CoA to result in 3-hydroxyisovaleryl-CoA and anacid. This reaction is schematically illustrated in FIG. 19.

Preferably, said acyl-CoA is acetyl-CoA.

Thus, the present invention also relates to a method for producing3-hydroxyisovaleryl-CoA from 3-hydroxyisovalerate (HIV) wherein3-hydroxyisovalerate reacts with an acyl-CoA, preferably withacetyl-CoA, to result in 3-hydroxyisovaleryl-CoA and a respective acid.

Preferably, this conversion is achieved by making use of an enzyme whichis classified as a CoA-transferase (EC 2.8.3.-). As regards thepreferred embodiments of said CoA-transferase (EC 2.8.3.-) in thecontext of step Xb, the same applies, mutatis mutandis, as has been setforth above with respect to the CoA-transferases (EC 2.8.3.-) in theenzymatic conversion of 3-hydroxyisovalerate (HIV) into 3-methylcrotonicacid with a concomitant transfer of CoA from 3-methylcrotonyl-CoA on3-hydroxyisovalerate (HIV) to result in 3-hydroxyisovaleryl-CoA (step Xaas shown in FIG. 18).

The Enzymatic Conversion of 3-Hydroxyisovaleryl-CoA into3-Methylcrotonyl-CoA as Shown in Step XI of FIG. 18

In addition or in the alternative to the above-described methods (stepVII), the 3-methylcrotonyl-CoA may be provided by an enzymatic reactionwherein 3-hydroxyisovaleryl-CoA is enzymatically converted into3-methylcrotonyl-CoA (step XI as shown in FIG. 18). This reversiblereaction is a dehydration reaction wherein 3-hydroxyisovaleryl-CoA isdehydrated into 3-methylcrotonyl-CoA and is schematically illustrated inFIG. 21.

Thus, the present invention also relates to a method for producingisobutene from 3-hydroxyisovaleryl-CoA wherein 3-hydroxyisovaleryl-CoAis first enzymatically converted into 3-methylcrotonyl-CoA wherein3-methylcrotonyl-CoA is further enzymatically converted into3-methylcrotonic acid according to any of the above-described methods.Further, the thus produced 3-methylcrotonic acid is then enzymaticallyconverted into isobutene as described herein above.

According to the present invention, the enzymatic conversion of3-hydroxyisovaleryl-CoA into 3-methylcrotonyl-CoA preferably makes useof

(i) an enoyl-CoA hydratase (EC 4.2.1.17);

(ii) a long-chain-enoyl-CoA hydratase (EC 4.2.1.74);

(iii) a 3-hydroxypropionyl-CoA dehydratase (EC 4.2.1.116);

(iv) a 3-hydroxybutyryl-CoA dehydratase (EC 4.2.1.55);

(v) a 3-hydroxyoctanoyl-[acyl-carrier-protein] dehydratase (EC4.2.1.59);

(vi) a crotonyl-[acyl-carrier-protein] hydratase (EC 4.2.1.58);

(vii) a 3-hydroxydecanoyl-[acyl-carrier-protein] dehydratase (EC4.2.1.60);

(viii) a 3-hydroxypalmitoyl-[acyl-carrier-protein] dehydratase (EC4.2.1.61); or

(ix) a 3-methylglutaconyl-CoA hydratase (EC 4.2.1.18).

In a preferred embodiment of the method according to the invention theconversion of 3-hydroxyisovaleryl-CoA into 3-methylcrotonyl-CoA isachieved by the use of an enoyl-CoA hydratase (EC 4.2.1.17). Enoyl-CoAhydratases (EC 4.2.1.17) as well as preferred enzymes of this enzymeclass have already been described above. Accordingly, as regards theseenzymes, the same applies to the conversion of 3-hydroxyisovaleryl-CoAinto 3-methylcrotonyl-CoA as has been set forth above.

In another preferred embodiment of the method according to the inventionthe conversion of 3-hydroxyisovaleryl-CoA into 3-methylcrotonyl-CoA isachieved by the use of a long-chain-enoyl-CoA hydratase (EC 4.2.1.74).Long-chain-enoyl-CoA hydratases (EC 4.2.1.74) catalyze the followingreaction:

(3S)-3-hydroxyacyl-CoA

trans-2-enoyl-CoA+H₂O

This enzyme belongs to the family of lyases, specifically thehydro-lyases, which cleave carbon-oxygen bonds. The systematic name ofthis enzyme class is long-chain-(3S)-3-hydroxyacyl-CoA hydro-lyase. Thisenzyme is also called long-chain enoyl coenzyme A hydratase and itparticipates in fatty acid elongation in mitochondria and fatty acidmetabolism. This enzyme occurs in a number of organisms, e.g., in Rattusnorvegicus (Wu et al., Org. Lett. 10 (2008), 2235-2238), Sus scrofa andCavia porcellus (Fong and Schulz, J. Biol. Chem. 252 (1977), 542-547;Schulz, Biol. Chem. 249 (1974), 2704-2709) and in principle anylong-chain-enoyl-CoA hydratase which can catalyze the conversion of3-hydroxyisovaleryl-CoA into 3-methylcrotonyl-CoA can be employed in themethod of the invention.

The Enzymatic Conversion of 3-Hydroxyisovalerate (HIV) into3-Hydroxyisovaleryl-CoA as Shown in Step XII of FIG. 18

In addition or in the alternative to the above-described methods (stepXa or step Xb), the 3-hydroxyisovaleryl-CoA may also be provided by anenzymatic conversion of 3-hydroxyisovalerate (HIV) into said3-hydroxyisovaleryl-CoA (step XII as shown in FIG. 18). This generalreaction wherein coenzyme A (CoASH) is fixed is schematicallyillustrated in FIG. 22.

Thus, the present invention also relates to a method for producingisobutene from 3-hydroxyisovalerate (HIV) in which 3-hydroxyisovalerate(HIV) is first converted into 3-hydroxyisovaleryl-CoA wherein3-hydroxyisovaleryl-CoA is then enzymatically converted into3-methylcrotonyl-CoA wherein 3-methylcrotonyl-CoA is furtherenzymatically converted into 3-methylcrotonic acid according to any ofthe above-described methods. Further, the thus produced 3-methylcrotonicacid is then enzymatically converted into isobutene as described hereinabove.

Moreover, the present invention also relates to a method for producing3-hydroxyisovaleryl-CoA from 3-hydroxyisovalerate (HIV).

According to the present invention, the enzymatic conversion of3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA preferably makesuse of an enzyme belonging to the family of ligases forming acarbon-sulfur bond (EC 6.2.1.-). The general reaction of the enzymaticconversion of 3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoAwherein coenzyme A (CoASH) is fixed can be catalyzed by an enzymebelonging to the family of ligases forming a carbon-sulfur bond (EC6.2.1.-) via two alternative mechanisms. In a first alternativereaction, an acyl-AMP is generated as an intermediate before coenzyme Ais fixed as schematically illustrated in FIG. 23.

In a second alternative reaction, an acyl phosphate is generated as anintermediate before coenzyme A is fixed as schematically illustrated inFIG. 24.

Enzymes which belong to the family of ligases forming a carbon-sulfurbond (EC 6.2.1.-) which are capable of enzymatically converting3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA wherein anacyl-AMP intermediate (i.e., the acyl adenylate intermediate3-hydroxyisovaleryl-adenosine monophosphate) is generated beforecoenzyme A is fixed coenzyme A (CoASH) share common structural motifswhich are referenced in the InterPro (InterPro44.0; Release Sep. 25,2013) as InterPro IPR020845, AMP-binding, conserved site(http://www.ebi.ac.uk/interpro/entry/IPR020845) and I PR000873(http://www.ebi.ac.uk/interpro/entry/IPR000873). The accession numberfor these enzymes in the Pfam database is PF00501.

As regards the first alternative reaction (wherein an acyl-AMP isgenerated as an intermediate before coenzyme A is fixed as schematicallyillustrated in FIG. 23), examples of enzymes which belong to the abovefamily of ligases forming a carbon-sulfur bond (EC 6.2.1.-) which arecapable of enzymatically converting 3-hydroxyisovalerate (HIV) into3-hydroxyisovaleryl-CoA wherein an acyl-AMP intermediate (i.e., the acyladenylate intermediate 3-hydroxyisovaleryl-adenosine monophosphate) isgenerated before coenzyme A is fixed coenzyme A (CoASH) and which may beused in the method for producing 3-hydroxyisovaleryl-CoA from3-hydroxyisovalerate (HIV) are summarized in the following Table A:

TABLE A CoA ligases (EC 6.2.1.-) capable of enzymatically converting 3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA involving anacyl-adenylate as an intermediate Enzyme name EC number Acetate-CoAligase 6.2.1.1 Butyrate-CoA ligase 6.2.1.2 Long chain fatty-acid-CoAligase 6.2.1.3 4-coumarate-CoA ligase 6.2.1.12 Arachidonate-CoA ligase6.2.1.15 Propionate-CoA ligase 6.2.1.17 Phytanate-CoA ligase 6.2.1.24o-succinylbenzoate-CoA ligase 6.2.1.26 3-alpha,7-alpha-dihydroxy-5-6.2.1.28 beta-cholestanate-CoA ligase 2-furoate-CoA ligase 6.2.1.314-chlorobenzoate-CoA ligase 6.2.1.33 3-hydroxybenzoate-CoA ligase6.2.1.37 4-hydroxybutyrate-CoA ligase 6.2.1.403-oxocholest-4-en-26-oate--CoA ligase 6.2.1.423-(methylthio)propionyl-CoA ligase 6.2.1.44 Cholate-CoA ligase 6.2.1.7Oxalate-CoA ligase 6.2.1.8 Biotin-CoA ligase 6.2.1.116-carboxyhexanoate-CoA ligase 6.2.1.14 Acetoacetate-CoA ligase 6.2.1.16Dicarboxylate-CoA ligase 6.2.1.23 Benzoate-CoA ligase 6.2.1.254-hydroxybenzoate-CoA ligase 6.2.1.27 Phenylacetate-CoA ligase 6.2.1.30Anthranilate-CoA ligase 6.2.1.32 3-hydroxypropionyl-CoA synthase6.2.1.36 (2,2,3-trimethy1-5-oxocyclopent-3- 6.2.1.38 enyl)acetyl-CoAsynthase 3-((3aS,4S,7aS)-7a-methy1-1,5-dioxo- 6.2.1.41octahydro-1H-inden-4-yl)propanoate- CoA ligase2-hydroxy-7-methoxy-5-methyl- 6.2.1.43 1-naphthoate-CoA ligaseMalonate-CoA ligase 6.2.1.n3

In a preferred embodiment, the enzymatic conversion of3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA via an acyladenylate intermediate can, e.g., be achieved by the use of abutanoate:CoA ligase (AMP forming) (EC 6.2.1.2). Butanoate:CoA ligasesare enzymes which catalyze the following reaction:

ATP+a carboxylate+CoA→AMP+diphosphate+an acyl-CoA

These enzymes participate in butanoate metabolism. The occurrence ofthese enzymes has been described for a large number of organisms,including prokaryotes and eukaryotes, in particular, bacteria, algae,fungi, plants and animals, e.g. for Methanobacterium formicum,Streptomyces coelicolor, Mycobacterium avium, Penicillium chrysogenum,Paecilomyces variotii, Pseudomonas aeruginosa, Dictyostelium discoideum,Cavia porcellus, Ovis aries, Sus scrofa, Bos taurus, Mus musculus,Rattus norvegicus, and Homo sapiens.

In a preferred embodiment, the enzymatic conversion of3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA via an acyladenylate intermediate is achieved by making use of a butanoate:CoAligase (AMP forming) (EC 6.2.1.2) derived from Methanobacteriumformicum. The amino acid sequence of said protein is shown in SEQ ID NO:77.

In a preferred embodiment of the present invention the butanoate:CoAligase (AMP forming) is an enzyme comprising the amino acid sequence ofSEQ ID NO: 77 or a sequence which is at least n % identical to SEQ IDNO: 77 with n being an integer between 10 and 100, preferably 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93,94, 95, 96, 97, 98 or 99 and wherein the enzyme has the enzymaticactivity of converting 3-hydroxyisovalerate (HIV) into3-hydroxyisovaleryl-CoA. As regards the determination of the sequenceidentity, the same applies as has been set forth above.

As regards the second alternative reaction (wherein an acyl phosphate isgenerated as an intermediate before coenzyme A is fixed as schematicallyillustrated in FIG. 24), examples of enzymes which belong to the abovefamily of ligases forming a carbon-sulfur bond (EC 6.2.1.-) which arecapable of enzymatically converting 3-hydroxyisovalerate (HIV) into3-hydroxyisovaleryl-CoA wherein an acyl phosphate intermediate (i.e.,the acyl phosphate intermediate 3-hydroxyisovaleryl phosphate) isgenerated before coenzyme A is fixed coenzyme A (CoASH) and which may beused in the method for producing 3-hydroxyisovaleryl-CoA from3-hydroxyisovalerate (HIV) are summarized in the following Table B.

TABLE B CoA ligases (EC 6.2.1.-) capable of enzymatically converting 3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA involving an acylphosphate as an intermediate Enzyme name EC number Succinate-CoA ligase(GDP-forming) 6.2.1.4 Glutarate-CoA ligase 6.2.1.6 Acid-CoA ligase(GDP-forming) 6.2.1 .10 Citrate-CoA ligase 6.2.1 .18 Succinate-CoAligase (ADP-forming) 6.2.1.5 Malate-CoA ligase 6.2.1.9 Acetate-CoAligase (ADP-forming) 6.2.1 .13

The Alternative Route for the Enzymatic Conversion from Acetyl-CoA intoIsobutene via 3-Methyl-3-Butenoyl-CoA and 3-Methyl-3-Butenoic Acid

In an alternative to the above, the present invention also relates to amethod for the production of isobutene via an alternative route as alsoshown in FIG. 1 wherein isobutene is produced by the enzymaticconversion of 3-methyl-3-butenoic acid into isobutene. Thus, the presentinvention provides a method for the production of isobutene comprisingthe enzymatic conversion of 3-methyl-3-butenoic acid into isobutene.Preferably, the enzymatic conversion of 3-methyl-3-butenoic acid intoisobutene is achieved by making use of an 3-methyl-3-butenoic aciddecarboxylase.

In accordance with this alternative route, the present invention notonly relates to a method for the production of isobutene from3-methyl-3-butenoic acid. Rather, as will be outlined in more detailfurther below, this conversion is preferably embedded in a pathway forthe production of isobutene starting from acetyl-CoA which is a centralcomponent and an important key molecule in metabolism used in manybiochemical reactions.

Therefore, the present invention also relates to a pathway starting fromacetyl-CoA wherein two acetyl-CoA molecules are enzymatically condensedinto acetoacetyl-CoA. Alternatively, acetyl-CoA is enzymaticallyconverted into malonyl-CoA which may then be converted into saidacetoacetyl-CoA by the enzymatic condensation of malonyl-CoA andacetyl-CoA into said acetoacetyl-CoA.

Further, the thus produced acetoacetyl-CoA can enzymatically beconverted into 3-methyl-3-butenoic acid (which is then ultimatelyconverted into isobutene) via the following briefly summarized pathway.

In this pathway, the thus produced acetoacetyl-CoA can furtherenzymatically be converted into 3-hydroxy-3-methylglutaryl-CoA.Moreover, the thus produced 3-hydroxy-3-methylglutaryl-CoA can furtherenzymatically be converted into 3-methylglutaconyl-CoA. Further, thethus produced 3-methylglutaconyl-CoA can enzymatically be converted into3-methyl-3-butenoyl-CoA. Further, the thus produced3-methyl-3-butenoyl-CoA can further be converted in a subsequentenzymatic reaction into 3-methyl-3-butenoic acid (which can thenultimately be converted into isobutene as described above and furtherbelow).

The Enzymatic Conversion of 3-Methyl-3-Butenoic Acid into Isobutene:Step XVI as Shown in FIG. 1

According to the present invention, the enzymatic conversion of3-methyl-3-butenoic acid into isobutene can be achieved by adecarboxylation. “Decarboxylation” is generally a chemical reaction thatremoves a carboxyl group and releases carbon dioxide (CO₂); see FIG. 25.

The enzymatic conversion of 3-methyl-3-butenoic acid into isobutene canpreferably be achieved by making use of an 3-methyl-3-butenoic aciddecarboxylase. In accordance with the present invention, an3-methyl-3-butenoic acid decarboxylase is an enzyme which is capable ofconverting 3-methyl-3-butenoic acid into isobutene. In preferredembodiments, the 3-methyl-3-butenoic acid decarboxylase is selected fromthe group consisting of:

-   -   (i) an FMN-dependent decarboxylase associated with an FMN prenyl        transferase; or    -   (ii) an aconitate decarboxylase (EC 4.1 .1.6); or    -   (iii) a methylcrotonyl-CoA carboxylase (EC 6.4.1.4); or    -   (iv) a geranoyl-CoA carboxylase (EC 6.4.1.5); or    -   (v) a protocatechuate (PCA) decarboxylase (EC 4.1.1.63).

In other preferred embodiments, the 3-methyl-3-butenoic aciddecarboxylase is selected from the group consisting of:6-methylsalicylate decarboxylase (EC 4.1.1.52), 2-oxo-3-hexenedioatedecarboxylase (EC 4.1.1 .77) and 5-oxopent-3-ene-1,2,5-tricarboxylatedecarboxylase (EC 4.1.1.68).

As regards the afore-mentioned embodiment, for the FMN-dependentdecarboxylase associated with an FMN prenyl transferase, the aconitatedecarboxylase (EC 4.1.1.6), the methylcrotonyl-CoA carboxylase (EC6.4.1.4), the geranoyl-CoA carboxylase (EC 6.4.1.5), the protocatechuate(PCA) decarboxylase (EC 4.1.1.63), the 6-methylsalicylate decarboxylase(EC 4.1.1.52), the 2-oxo-3-hexenedioate decarboxylase (EC 4.1.1.77) andthe 5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylase (EC 4.1.1.68),the same applies as has been set forth above in connection with othermethods of the present invention.

The Enzymatic Conversion of 3-Methyl-3-Butenoyl-CoA into3-Methyl-3-Butenoic Acid: Steps XVIIa, XVIIb or XVIIc as Shown in FIG. 1

The 3-methyl-3-butenoic acid may itself be provided by an enzymaticreaction, namely the enzymatic conversion of 3-methyl-3-butenoyl-CoAinto 3-methyl-3-butenoic acid; see FIG. 26.

Accordingly, the present invention relates to a method for producingisobutene from 3-methyl-3-butenoyl-CoA in which 3-methyl-3-butenoyl-CoAis first converted into 3-methyl-3-butenoic acid which is then furtherconverted into isobutene as described herein above.

According to the present invention, the conversion of3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoic acid can, e.g., beachieved by three different alternative enzymatic routes, i.e., by:

-   -   (a) a single enzymatic reaction (see FIG. 27) in which        3-methyl-3-butenoyl-CoA is directly converted into        3-methyl-3-butenoic acid, preferably by making use of a CoA        transferase (EC 2.8.3.-), preferably a propionate:acetate-CoA        transferase (EC 2.8.3.1), an acetate CoA-transferase (EC        2.8.3.8) or a succinyl-CoA:acetate CoA-transferase (EC        2.8.3.18);    -   (b) a single enzymatic reaction(see FIG. 28) in which        3-methyl-3-butenoyl-CoA is directly converted into        3-methyl-3-butenoic acid, preferably by making use of a        thioester hydrolase (EC 3.1.2.-), preferably acetyl-CoA        hydrolase (EC 3.1.2.1), an ADP-dependent short-chain-acyl-CoA        hydrolase (EC 3.1.2.18) or an acyl-CoA hydrolase (EC 3.1.2.20);        or    -   (c) two enzymatic steps (see FIG. 29) comprising        -   (i) first enzymatically converting 3-methyl-3-butenoyl-CoA            into 3-methyl-3-butenoyl phosphate, preferably by making use            of a phosphate butyryltransferase (EC 2.3.1.19) or a            phosphate acetyltransferase (EC 2.3.1.8); and        -   (ii) then enzymatically converting the thus obtained            3-methyl-3-butenoyl phosphate into said 3-methyl-3-butenoic            acid, preferably by making use of a phosphotransferase with            a carboxy group as acceptor (EC 2.7.2.-), preferably a            propionate kinase (EC 2.7.2.15), an acetate kinase (EC            2.7.2.1), a butyrate kinase (EC 2.7.2.7) or a            branched-chain-fatty-acid kinase (EC 2.7.2.14).

As regards the aforementioned embodiments, for the CoA transferase (EC2.8.3.-), the propionate:acetate-CoA transferase (EC 2.8.3.1), theacetate CoA-transferase (EC 2.8.3.8), the succinyl-CoA:acetateCoA-transferase (EC 2.8.3.18), the thioester hydrolase (EC 3.1.2.-), theacetyl-CoA hydrolase (EC 3.1.2.1), the ADP-dependentshort-chain-acyl-CoA hydrolase (EC 3.1.2.18), the an acyl-CoA hydrolase(EC 3.1.2.20) the phosphate butyryltransferase (EC 2.3.1.19), thephosphate acetyltransferase (EC 2.3.1.8), the phosphotransferase with acarboxy group as acceptor (EC 2.7.2.-), the propionate kinase (EC2.7.2.15), the acetate kinase (EC 2.7.2.1), the butyrate kinase (EC2.7.2.7) and the branched-chain-fatty-acid kinase (EC 2.7.2.14), thesame applies as has been set forth above in connection with the othermethods of the present invention.

The Enzymatic Conversion of 3-Methylglutaconyl-CoA into3-Methyl-3-Butenoyl-CoA: Step XVIII as Shown in FIG. 1

The 3-methyl-3-butenoyl-CoA may itself be provided by an enzymaticreaction, namely the enzymatic conversion of 3-methylglutaconyl-CoA into3-methyl-3-butenoyl-CoA; see FIG. 30.

Accordingly, the present invention relates to a method for producingisobutene from 3-methyl-3-butenoyl-CoA in which 3-methylglutaconyl-CoAis first converted into 3-methyl-3-butenoyl-CoA which is then furtherconverted into 3-methyl-3-butenoic acid which is then further convertedinto isobutene as described herein above.

Moreover, the present invention relates to a method for producing3-methyl-3-butenoyl-CoA by converting 3-methylglutaconyl-CoA into3-methyl-3-butenoyl-CoA.

According to the present invention, the conversion of3-methylglutaconyl-CoA into 3-methyl-3-butenoyl-CoA can preferably beachieved by making use of

-   -   (a) (i) a methylcrotonyl-CoA carboxylase (EC 6.4.1.4); or (ii) a        geranoyl-CoA carboxylase (EC 6.4.1.5),    -   (b) an N-terminal domain of CurF from Lynbya majuscula        multifunctional protein or a 3-methylglutaconyl-CoA        decarboxylase, preferably a 3-methylglutaconyl-CoA decarboxylase        of Myxococcus xanthus encoded by the liuB gene; or    -   (c) an enzyme of the 4-oxalocrotonate decarboxylase family.

As regards the aforementioned embodiments, for the methylcrotonyl-CoAcarboxylase (EC 6.4.1.4), the geranoyl-CoA carboxylase (EC 6.4.1.5) andthe 3-methylglutaconyl-CoA decarboxylase, preferably the3-methylglutaconyl-CoA decarboxylase of Myxococcus xanthus encoded bythe liuB gene, the same applies as has been set forth above inconnection with the other methods of the present invention.

In a preferred embodiment the conversion of 3-methylglutaconyl-CoA viadecarboxylation into 3-methyl-3-butenoyl-CoA is catalyzed by anN-terminal domain of CurF from Lynbya majuscula multifunctional protein.The N-terminal domain of CurF from Lynbya majuscula multifunctionalprotein is a domain of a polyketide synthase (PKS)/non ribosomalepeptide synthase (NRPS) of the CurF multifunctional protein from Lyngbyamajuscula. This N-terminal domain of CurF has been classified as aprotein belonging to the crotonase superfamily by studying the crystalstructure and it naturally catalyzes the decarboxylation of3-methylglutaconyl-ACP (Acyl Carrier Protein) into3-methyl-crotonyl-ACP. ACP is similar to CoA as both molecules have aphosphopantetheine moiety in common (as shown in FIG. 31). Moreover,both ACP and CoA can form a thioester with a biological acid (J. Biol.Chem. 289: 35957-35963 (2007) and Chemistry & Biology 11:817-833(2004)).

In another preferred embodiment the conversion of 3-methylglutaconyl-CoAvia decarboxylation into 3-methyl-3-butenoyl-CoA is catalyzed by anenzyme of the 4-oxalocrotonate decarboxylase family (EC 4.1.1.77).

4-oxalocrotonate decarboxylases (EC 4.1.1.77) catalyse the followingreaction:

(3E)-2-oxohex-3-enedioate

2-oxopent-4-enoate+CO2

This enzyme is known from various organisms and has, e.g., beendescribed in Bortetella sp., Cupriavidus nector, Geobacillusstearothermophilus, Pseudomonas putida and Ralstonia pickettii. Thus, ina preferred embodiment, the 4-oxalocrotonate decarboxylase used for theconversion of 3-methylglutaconyl-CoA via decarboxylation into3-methyl-3-butenoyl-CoA is a 4-oxalocrotonate decarboxylase derived fromgenus Bortetella, Cupriavidus, Geobacillus, Pseudomonas pr Ralstonia,more preferably from the species Bortetella sp., Cupriavidus nector,Geobacillus stearothermophilus, Pseudomonas putida or Ralstoniapickettii. In an even more preferred embodiment, the 4-oxalocrotonatedecarboxylase used for the conversion of 3-methylglutaconyl-CoA viadecarboxylation into 3-methyl-3-butenoyl-CoA is the 4-oxalocrotonatedecarboxylase of Geobacillus stearothermophilus (UniProt Accessionnumber BOVXM8).

In a preferred embodiment, the 4-oxalocrotonate decarboxylase employedin the method of the present invention in the conversion of3-methylglutaconyl-CoA via decarboxylation into 3-methyl-3-butenoyl-CoAis derived from Geobacillus stearothermophilus and has an amino acidsequence as shown SEQ ID NO:69.

In a preferred embodiment of the present invention the 4-oxalocrotonatedecarboxylase is an enzyme comprising the amino acid sequence of SEQ IDNO: 69 or a sequence which is at least n % identical to SEQ ID NO: 69with n being an integer between 10 and 100, preferably 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95,96, 97, 98 or 99 and wherein the enzyme has the enzymatic activity ofconverting 3-methylglutaconyl-CoA via decarboxylation into3-methyl-3-butenoyl-CoA. As regards the determination of the sequenceidentity, the same applies as has been set forth above.

The Enzymatic Conversion of 3-Hydroxy-3-Methylglutaryl-CoA into3-Methylglutaconyl-CoA: Step VIII as Shown in FIG. 1

The 3-methylglutaconyl-CoA which can be converted into3-methyl-3-butenoyl-CoA according to any of the above described methodsmay itself be provided by an enzymatic reaction, namely the enzymaticconversion of 3-hydroxy-3-methylglutaryl-CoA into3-methylglutaconyl-CoA.

Accordingly, the present invention also relates to a method forproducing isobutene from 3-hydroxy-3-methylglutaryl-CoA in which3-hydroxy-3-methylglutaryl-CoA is first converted into3-methylglutaconyl-CoA which is then converted into3-methyl-3-butenoyl-CoA which is then further converted into3-methyl-3-butenoic acid which is then further converted into isobuteneas described herein above.

According to the present invention, the enzymatic conversion of3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA is anenzymatic dehydration reaction which occurs naturally, and which iscatalyzed, e.g., by enzymes classified as 3-methylglutaconyl-coenzyme Ahydratase (EC 4.2.1.18). Accordingly, the enzymatic conversion of3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA preferablymakes use of a 3-methylglutaconyl-coenzyme A hydratase (EC 4.2.1.18).

As regards the afore-mentioned embodiment, for the enzymes classified as3-methylglutaconyl-coenzyme A hydratase (EC 4.2.1.18), the same appliesas has been set forth above in connection with the other methods of thepresent invention.

The Enzymatic Conversion of Acetoacetyl-CoA into3-Hydroxy-3-Methylglutaryl-CoA: Step IX as Shown in FIG. 1

The 3-hydroxy-3-methylglutaryl-CoA may itself be provided by anenzymatic reaction, namely the enzymatic condensation of acetoacetyl-CoAand acetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA which has alreadybeen described in detail above.

Accordingly, the present invention also relates to a method forproducing isobutene from acetoacetyl-CoA and acetyl-CoA in whichacetoacetyl-CoA and acetyl-CoA are first condensed into3-hydroxy-3-methylglutaryl-CoA which is then converted into3-methylglutaconyl-CoA which is then converted into3-methyl-3-butenoyl-CoA which is then further converted into3-methyl-3-butenoic acid which is then further converted into isobuteneas described herein above.

The Enzymatic Conversion of Acetyl-CoA into Acetoacetyl-CoA: Step XIII,Step XIV and Step XV as Shown in FIG. 1

The acetoacetyl-CoA may itself be provided by an enzymatic reaction,namely the enzymatic conversion of acetyl-CoA into acetoacetyl-CoA viaseveral different routes which have already been described in detailabove.

Thus, the present invention also relates to a method for producingisobutene from acetyl-CoA in which acetyl-CoA is first converted intoacetoacetyl-CoA by any of the above-mentioned routes which is thencondensed with acetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA which isthen converted into 3-methylglutaconyl-CoA which is then converted into3-methyl-3-butenoyl-CoA which is then further converted into3-methyl-3-butenoic acid which is then further converted into isobuteneas described herein above.

Summarizing the alternative route for the enzymatic conversion fromacetyl-CoA into isobutene via 3-methyl-3-butenoyl-CoA and3-methyl-3-butenoic acid as outlined above, the present invention alsorelates to the following embodiments as characterized by the followingitems 1 to 26:

-   -   1. A method for the production of isobutene comprising the        enzymatic conversion of 3-methyl-3-butenoic acid into isobutene.    -   2. The method of item 1, wherein the enzymatic conversion of        3-methyl-3-butenoic acid into isobutene is achieved by making        use of an 3-methyl-3-butenoic acid decarboxylase.    -   3. The method of item 2, wherein the 3-methyl-3-butenoic acid        decarboxylase is:        -   (i) an FMN-dependent decarboxylase associated with an FMN            prenyl transferase; or        -   (ii) an aconitate decarboxylase (EC 4.1.1.6); or        -   (iii) a methylcrotonyl-CoA carboxylase (EC 6.4.1.4); or        -   (iv) a geranoyl-CoA carboxylase (EC 6.4.1.5); or        -   (v) a protocatechuate (PCA) decarboxylase (EC 4.1.1.63).    -   4. The method of item 1 or 2, further comprising providing the        3-methyl-3-butenoic acid by the enzymatic conversion of        3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoic acid.    -   5. The method of item 4, wherein the enzymatic conversion of        3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoic acid is        achieved by        -   (a) a single enzymatic reaction in which            3-methyl-3-butenoyl-CoA is directly converted into            3-methyl-3-butenoic acid by making use of a CoA transferase            (EC 2.8.3.-), preferably a propionate:acetate-CoA            transferase (EC 2.8.3.1), an acetate CoA-transferase (EC            2.8.3.8) or a succinyl-CoA:acetate CoA-transferase (EC            2.8.3.18);        -   (b) a single enzymatic reaction in which            3-methyl-3-butenoyl-CoA is directly converted into            3-methyl-3-butenoic acid by making use of a thioester            hydrolase (EC 3.1.2.-), preferably acetyl-CoA hydrolase (EC            3.1.2.1), an ADP-dependent short-chain-acyl-CoA hydrolase            (EC 3.1.2.18) or an acyl-CoA hydrolase (EC 3.1.2.20);        -   (c) two enzymatic steps comprising            -   (i) first enzymatically converting                3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoyl                phosphate; and            -   (ii) then enzymatically converting the thus obtained                3-methyl-3-butenoyl phosphate into said                3-methyl-3-butenoic acid.    -   6. The method of item 5(c), wherein the enzymatic conversion of        said 3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoyl phosphate        is achieved by making use of a phosphate butyryltransferase (EC        2.3.1.19) or a phosphate acetyltransferase (EC 2.3.1.8) and the        enzymatic conversion of said 3-methyl-3-butenoyl phosphate into        said 3-methyl-3-butenoic acid is achieved by making use of a        phosphotransferase with a carboxy group as acceptor (EC        2.7.2.-), preferably a propionate kinase (EC 2.7.2.15), an        acetate kinase (EC 2.7.2.1), a butyrate kinase (EC 2.7.2.7) or a        branched-chain-fatty-acid kinase (EC 2.7.2.14).    -   7. The method of any one of items 1 to 4, further comprising        providing the 3-methyl-3-butenoyl-CoA by the enzymatic        conversion of 3-methylglutaconyl-CoA into        3-methyl-3-butenoyl-CoA.    -   8. The method of item 7, wherein the enzymatic conversion of        3-methylglutaconyl-CoA into 3-methyl-3-butenoyl-CoA is achieved        by making use of        -   (a) (i) a methylcrotonyl-CoA carboxylase (EC 6.4.1.4);            or (ii) a geranoyl-CoA carboxylase (EC 6.4.1.5),        -   (b) an N-terminal domain of CurF from Lynbya majuscula            multifunctional protein or a 3-methylglutaconyl-CoA            decarboxylase, preferably a 3-methylglutaconyl-CoA            decarboxylase of Myxococcus xanthus encoded by the liuB            gene; or        -   (c) an enzyme of the 4-oxalocrotonate decarboxylase family.    -   9. The method of any one of items 1 to 8, further comprising        providing the 3-methylglutaconyl-CoA by the enzymatic conversion        of 3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA.    -   10. The method of item 9, wherein the enzymatic conversion of        3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA is        achieved by making use of a 3-methylglutaconyl-coenzyme A        hydratase (EC 4.2.1.18), a 3-hydroxyacyl-CoA dehydratase (EC        4.2.1.-) or an enoyl-CoA hydratase (EC 4.2.1.-).    -   11. The method of any one of items 1 to 10, further comprising        providing the 3-hydroxy-3-methylglutaryl-CoA by the enzymatic        condensation of acetoacetyl-CoA and acetyl-CoA into        3-hydroxy-3-methylglutaryl-CoA.    -   12. The method of item 11, wherein the enzymatic condensation of        acetoacetyl-CoA and acetyl-CoA into        3-hydroxy-3-methylglutaryl-CoA is achieved by making use of a        3-hydroxy-3-methylglutaryl-CoA synthase.    -   13. The method of any one of items 1 to 12, further comprising        providing the acetoacetyl-CoA by the enzymatic conversion of        acetyl-CoA into acetoacetyl-CoA comprising:        -   (a) two enzymatic steps comprising            -   (i) first the enzymatic conversion of acetyl-CoA into                malonyl-CoA; and            -   (ii) then enzymatically condensing the thus obtained                malonyl-CoA and acetyl-CoA into said acetoacetyl-CoA; or        -   (b) a single enzymatic reaction in which two molecules of            acetyl-CoA are directly condensed into acetoacetyl-CoA.    -   14. The method of item 13(a)(i), wherein the enzymatic        conversion of acetyl-CoA into malonyl-CoA is achieved by making        use of an acetyl-CoA carboxylase (EC 6.4.1.2).    -   15. The method of item 13(a)(ii), wherein the enzymatic        condensation of malonyl-CoA and acetyl-CoA into said        acetoacetyl-CoA is achieved by making use of an acetoacetyl-CoA        synthase (EC 2.3.1.194).    -   16. The method of item 13(b), wherein the direct enzymatic        condensation of two molecules of acetyl-CoA into acetoacetyl-CoA        is achieved by making use of an acetyl-CoA C-acetyltransferase        (EC 2.3.1.9).    -   17. A recombinant organism or microorganism which expresses        -   (i) an enzyme as defined in any one of items 1 to 3; and        -   (ii) an enzyme as defined in any one of items 4 to 6.    -   18. The recombinant organism or microorganism of item 17,        further expressing an enzyme as defined in item 7 or 8.    -   19. The recombinant organism or microorganism of item 18,        further expressing an enzyme as defined in item 9 or 10.    -   20. The recombinant organism or microorganism of item 19,        further expressing an enzyme as defined in item 11 or 12.    -   21. The recombinant organism or microorganism of item 20,        further expressing an enzyme as defined in claim 13.    -   22. The recombinant organism or microorganism of item 21,        further expressing an enzyme as defined in any one of claims 14        to 16.    -   23. Use of a recombinant organism or microorganism as defined in        any one of items 17 to 22 for the production of isobutene.    -   24. The use of a recombinant organism or microorganism of item        23, wherein said recombinant organism or microorganism expresses        an enzyme catalyzing the enzymatic conversion of        3-methyl-3-butenoic acid into isobutene.    -   25. Use of an enzyme catalyzing the enzymatic conversion of        3-methyl-3-butenoic acid into isobutene for the production of        isobutene from 3-methyl-3-butenoic acid.    -   26. A composition comprising 3-methyl-3-butenoic acid and a        recombinant organism or microorganism as defined in any one of        items 17 to 22; or 3-methyl-3-butenoic acid and an enzyme as        defined in any one of items 1 to 16.

A method according to the present invention may be carried out in vitroor in vivo. An in vitro reaction is understood to be a reaction in whichno cells are employed, i.e. an acellular reaction. Thus, in vitropreferably means in a cell-free system. The term “in vitro” in oneembodiment means in the presence of isolated enzymes (or enzyme systemsoptionally comprising possibly required cofactors). In one embodiment,the enzymes employed in the method are used in purified form.

For carrying out the method in vitro the substrates for the reaction andthe enzymes are incubated under conditions (buffer, temperature,cosubstrates, cofactors etc.) allowing the enzymes to be active and theenzymatic conversion to occur. The reaction is allowed to proceed for atime sufficient to produce the respective product. The production of therespective products can be measured by methods known in the art, such asgas chromatography possibly linked to mass spectrometry detection.

The enzymes may be in any suitable form allowing the enzymatic reactionto take place. They may be purified or partially purified or in the formof crude cellular extracts or partially purified extracts. It is alsopossible that the enzymes are immobilized on a suitable carrier.

In another embodiment the method according to the invention is carriedout in culture, in the presence of an organism, preferably amicroorganism, producing the enzymes described above for the conversionsof the methods according to the present invention as described hereinabove. A method which employs a microorganism for carrying out a methodaccording to the invention is referred to as an “in vivo” method. It ispossible to use a microorganism which naturally produces the enzymesdescribed above for the conversions of the methods according to thepresent invention or a microorganism which had been genetically modifiedso that it expresses (including overexpresses) one or more of suchenzymes. Thus, the microorganism can be an engineered microorganismwhich expresses enzymes described above for the conversions of themethods according to the present invention, i.e. which has in its genomea nucleotide sequence encoding such enzymes and which has been modifiedto overexpress them. The expression may occur constitutively or in aninduced or regulated manner.

In another embodiment the microorganism can be a microorganism which hasbeen genetically modified by the introduction of one or more nucleicacid molecules containing nucleotide sequences encoding one or moreenzymes described above for the conversions of the methods according tothe present invention. The nucleic acid molecule can be stablyintegrated into the genome of the microorganism or may be present in anextrachromosomal manner, e.g. on a plasmid.

Such a genetically modified microorganism can, e.g., be a microorganismthat does not naturally express enzymes described above for theconversions of the methods according to the present invention and whichhas been genetically modified to express such enzymes or a microorganismwhich naturally expresses such enzymes and which has been geneticallymodified, e.g. transformed with a nucleic acid, e.g. a vector, encodingthe respective enzyme(s), and/or insertion of a promoter in front of theendogenous nucleotide sequence encoding the enzyme in order to increasethe respective activity in said microorganism.

However, the invention preferably excludes naturally occurringmicroorganisms as found in nature expressing an enzyme as describedabove at levels as they exist in nature. Instead, the microorganism ofthe present invention and employed in a method of the present inventionis preferably a non-naturally occurring microorganism, whether it hasbeen genetically modified to express (including overexpression) anexogenous enzyme of the invention not normally existing in its genome orwhether it has been engineered to overexpress an exogenous enzyme.

Thus, the enzymes and (micro)organisms employed in connection with thepresent invention are preferably non-naturally occurring enzymes or(micro)organisms, i.e. they are enzymes or (micro)organisms which differsignificantly from naturally occurring enzymes or microorganism andwhich do not occur in nature. As regards the enzymes, they arepreferably variants of naturally occurring enzymes which do not as suchoccur in nature. Such variants include, for example, mutants, inparticular prepared by molecular biological methods, which show improvedproperties, such as a higher enzyme activity, higher substratespecificity, higher temperature resistance and the like. As regards the(micro)organisms, they are preferably genetically modified organisms asdescribed herein above which differ from naturally occurring organismsdue to a genetic modification. Genetically modified organisms areorganisms which do not naturally occur, i.e., which cannot be found innature, and which differ substantially from naturally occurringorganisms due to the introduction of a foreign nucleic acid molecule.

By overexpressing an exogenous or endogenous enzyme as described hereinabove, the concentration of the enzyme is substantially higher than whatis found in nature, which can then unexpectedly force the reaction ofthe present invention which uses a non-natural for the respectiveenzyme. Preferably, the concentration of the overexpressed enzyme is atleast 5%, 10%, 20%, 30% or 40% of the total host cell protein.

A “non-natural” substrate is understood to be a molecule that is notacted upon by the respective enzyme in nature, even though it mayactually coexist in the microorganism along with the endogenous enzyme.This “non-natural” substrate is not converted by the microorganism innature as other substrates are preferred (e.g. the “natural substrate”).Thus, the present invention contemplates utilizing a non-naturalsubstrate with the enzymes described above in an environment not foundin nature.

Thus, it is also possible in the context of the present invention thatthe microorganism is a microorganism which naturally does not have therespective enzyme activity but which is genetically modified so as tocomprise a nucleotide sequence allowing the expression of acorresponding enzyme. Similarly, the microorganism may also be amicroorganism which naturally has the respective enzyme activity butwhich is genetically modified so as to enhance such an activity, e.g. bythe introduction of an exogenous nucleotide sequence encoding acorresponding enzyme or by the introduction of a promoter for theendogenous gene encoding the enzyme to increase endogenous production tooverexpressed (non-natural) levels.

If a microorganism is used which naturally expresses a correspondingenzyme, it is possible to modify such a microorganism so that therespective activity is overexpressed in the microorganism. This can,e.g., be achieved by effecting mutations in the promoter region of thecorresponding gene or introduction of a high expressing promoter so asto lead to a promoter which ensures a higher expression of the gene.Alternatively, it is also possible to mutate the gene as such so as tolead to an enzyme showing a higher activity.

By using microorganisms which express enzymes described above for theconversions of the methods according to the present invention, it ispossible to carry out the methods according to the invention directly inthe culture medium, without the need to separate or purify the enzymes.

In one embodiment the organism employed in a method according to theinvention is a microorganism which has been genetically modified tocontain a foreign nucleic acid molecule encoding at least one enzymedescribed above for the conversions of the methods according to thepresent invention. The term “foreign” or “exogenous” in this contextmeans that the nucleic acid molecule does not naturally occur in saidmicroorganism. This means that it does not occur in the same structureor at the same location in the microorganism. In one preferredembodiment, the foreign nucleic acid molecule is a recombinant moleculecomprising a promoter and a coding sequence encoding the respectiveenzyme in which the promoter driving expression of the coding sequenceis heterologous with respect to the coding sequence. “Heterologous” inthis context means that the promoter is not the promoter naturallydriving the expression of said coding sequence but is a promoternaturally driving expression of a different coding sequence, i.e., it isderived from another gene, or is a synthetic promoter or a chimericpromoter. Preferably, the promoter is a promoter heterologous to themicroorganism, i.e. a promoter which does naturally not occur in therespective microorganism. Even more preferably, the promoter is aninducible promoter. Promoters for driving expression in different typesof organisms, in particular in microorganisms, are well known to theperson skilled in the art.

In a further embodiment the nucleic acid molecule is foreign to themicroorganism in that the encoded enzyme is not endogenous to themicroorganism, i.e. is naturally not expressed by the microorganism whenit is not genetically modified. In other words, the encoded enzyme isheterologous with respect to the microorganism. The foreign nucleic acidmolecule may be present in the microorganism in extrachromosomal form,e.g. as a plasmid, or stably integrated in the chromosome. A stableintegration is preferred. Thus, the genetic modification can consist,e.g. in integrating the corresponding gene(s) encoding the enzyme(s)into the chromosome, or in expressing the enzyme(s) from a plasmidcontaining a promoter upstream of the enzyme-coding sequence, thepromoter and coding sequence preferably originating from differentorganisms, or any other method known to one of skill in the art.

The term “microorganism” in the context of the present invention refersto bacteria, as well as to fungi, such as yeasts, and also to algae andarchaea. In one preferred embodiment, the microorganism is a bacterium.In principle any bacterium can be used. Preferred bacteria to beemployed in the process according to the invention are bacteria of thegenus Bacillus, Clostridium, Corynebacterium, Pseudomonas, Zymomonas orEscherichia. In a particularly preferred embodiment the bacteriumbelongs to the genus Escherichia and even more preferred to the speciesEscherichia coli. In another preferred embodiment the bacterium belongsto the species Pseudomonas putida or to the species Zymomonas mobilis orto the species Corynebacterium glutamicum or to the species Bacillussubtilis.

It is also possible to employ an extremophilic bacterium such as Thermusthermophilus, or anaerobic bacteria from the family Clostridiae.

In another preferred embodiment the microorganism is a fungus, morepreferably a fungus of the genus Saccharomyces, Schizosaccharomyces,Aspergillus, Trichoderma, Kluyveromyces or Pichia and even morepreferably of the species Saccharomyces cerevisiae, Schizosaccharomycespombe, Aspergillus niger, Trichoderma reesei, Kluyveromyces marxianus,Kluyveromyces lactis, Pichia pastoris, Pichia torula or Pichia utilis.

In another embodiment, the method according to the invention makes useof a photosynthetic microorganism expressing at least one enzyme for theconversion according to the invention as described above. Preferably,the microorganism is a photosynthetic bacterium, or a microalgae. In afurther embodiment the microorganism is an algae, more preferably analgae belonging to the diatomeae.

It is also conceivable to use in the method according to the invention acombination of microorganisms wherein different microorganisms expressdifferent enzymes as described above. The genetic modification ofmicroorganisms to express an enzyme of interest will also be furtherdescribed in detail below.

In a preferred embodiment, the method of the present invention makes useof an organism, preferably a microorganism, which is geneticallymodified in order to avoid the leakage of acetyl-CoA, thereby increasingthe intracellular concentration of acetyl-CoA. Genetic modificationsleading to an increase in the intracellular concentration of acetyl-CoAare known in the art. Without being bound to theory, such an organism,preferably a microorganism, may preferably be genetically modified bydeleting or inactivating the following genes:

ΔackA (acetate kinase), Δldh (lactate dehydrogenase), ΔadhE (alcoholdehydrogenase), ΔfrdB and/or ΔfrdC (fumarate reductase and fumaratedehydrogenase).

Alternatively, or in addition to any of the above deletions, theorganism or microorganism may genetically be modified by overexpressingthe gene panK/coaA encoding Pantothenate kinase, thereby increasing theCoA/acetyl-CoA intracellular pool.

These modifications which avoid the leakage of acetyl-CoA are known inthe art and corresponding modified organisms have been used in methodsfor the bioconversion of exogenous isoamyl alcohol into isoamyl acetateby an E. coli strain expressing ATF2 (Metab. Eng. 6 (2004), 294-309).

In another embodiment, the method of the invention comprises the step ofproviding the organism, preferably the microorganism carrying therespective enzyme activity or activities in the form of a (cell)culture, preferably in the form of a liquid cell culture, a subsequentstep of cultivating the organism, preferably the microorganism in afermenter (often also referred to a bioreactor) under suitableconditions allowing the expression of the respective enzyme and furthercomprising the step of effecting an enzymatic conversion of a method ofthe invention as described herein above. Suitable fermenter orbioreactor devices and fermentation conditions are known to the personskilled in the art. A bioreactor or a fermenter refers to anymanufactured or engineered device or system known in the art thatsupports a biologically active environment. Thus, a bioreactor or afermenter may be a vessel in which a chemical/biochemical like themethod of the present invention is carried out which involves organisms,preferably microorganisms and/or biochemically active substances, i.e.,the enzyme(s) described above derived from such organisms or organismsharbouring the above described enzyme(s). In a bioreactor or afermenter, this process can either be aerobic or anaerobic. Thesebioreactors are commonly cylindrical, and may range in size from litresto cubic metres, and are often made of stainless steel. In this respect,without being bound by theory, the fermenter or bioreactor may bedesigned in a way that it is suitable to cultivate the organisms,preferably microorganisms, in, e.g., a batch-culture,feed-batch-culture, perfusion culture or chemostate-culture, all ofwhich are generally known in the art.

The culture medium can be any culture medium suitable for cultivatingthe respective organism or microorganism.

In a preferred embodiment the method according to the present inventionalso comprises the step of recovering the isobutene produced by themethod. For example, if the method according to the present invention iscarried out in vivo by fermenting a corresponding microorganismexpressing the necessary enzymes, the isobutene can be recovered fromthe fermentation off-gas by methods known to the person skilled in theart.

In a preferred embodiment, the present invention relates to a method asdescribed herein above in which a microorganism as described hereinabove is employed, wherein the microorganism is capable of enzymaticallyconverting 3-methylcrotonic acid into isobutene, wherein said methodcomprises culturing the microorganism in a culture medium.

The enzymes used in the method according to the invention can benaturally occurring enzymes or enzymes which are derived from anaturally occurring enzymes, e.g. by the introduction of mutations orother alterations which, e.g., alter or improve the enzymatic activity,the stability, etc.

Methods for modifying and/or improving the desired enzymatic activitiesof proteins are well-known to the person skilled in the art and include,e.g., random mutagenesis or site-directed mutagenesis and subsequentselection of enzymes having the desired properties or approaches of theso-called “directed evolution”.

For example, for genetic modification in prokaryotic cells, a nucleicacid molecule encoding a corresponding enzyme can be introduced intoplasmids which permit mutagenesis or sequence modification byrecombination of DNA sequences. Standard methods (see Sambrook andRussell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, ColdSpring Harbor, N.Y., USA) allow base exchanges to be performed ornatural or synthetic sequences to be added. DNA fragments can be ligatedby using adapters and linkers complementary to the fragments. Moreover,engineering measures which provide suitable restriction sites or removesurplus DNA or restriction sites can be used. In those cases, in whichinsertions, deletions or substitutions are possible, in vitromutagenesis, “primer repair”, restriction or ligation can be used. Ingeneral, a sequence analysis, restriction analysis and other methods ofbiochemistry and molecular biology are carried out as analysis methods.The resulting enzyme variants are then tested for the desired activity,e.g., enzymatic activity, with an assay as described above and inparticular for their increased enzyme activity.

As described above, the microorganism employed in a method of theinvention or contained in the composition of the invention may be amicroorganism which has been genetically modified by the introduction ofa nucleic acid molecule encoding a corresponding enzyme. Thus, in apreferred embodiment, the microorganism is a recombinant microorganismwhich has been genetically modified to have an increased activity of atleast one enzyme described above for the conversions of the methodaccording to the present invention. This can be achieved e.g. bytransforming the microorganism with a nucleic acid encoding acorresponding enzyme. A detailed description of genetic modification ofmicroorganisms will be given further below. Preferably, the nucleic acidmolecule introduced into the microorganism is a nucleic acid moleculewhich is heterologous with respect to the microorganism, i.e. it doesnot naturally occur in said microorganism.

In the context of the present invention, an “increased activity” meansthat the expression and/or the activity of an enzyme in the geneticallymodified microorganism is at least 10%, preferably at least 20%, morepreferably at least 30% or 50%, even more preferably at least 70% or 80%and particularly preferred at least 90% or 100% higher than in thecorresponding non-modified microorganism. In even more preferredembodiments the increase in expression and/or activity may be at least150%, at least 200% or at least 500%. In particularly preferredembodiments the expression is at least 10-fold, more preferably at least100-fold and even more preferred at least 1000-fold higher than in thecorresponding non-modified microorganism.

The term “increased” expression/activity also covers the situation inwhich the corresponding non-modified microorganism does not express acorresponding enzyme so that the corresponding expression/activity inthe non-modified microorganism is zero. Preferably, the concentration ofthe overexpressed enzyme is at least 5%, 10%, 20%, 30%, or 40% of thetotal host cell protein.

Methods for measuring the level of expression of a given protein in acell are well known to the person skilled in the art. In one embodiment,the measurement of the level of expression is done by measuring theamount of the corresponding protein. Corresponding methods are wellknown to the person skilled in the art and include Western Blot, ELISAetc. In another embodiment the measurement of the level of expression isdone by measuring the amount of the corresponding RNA. Correspondingmethods are well known to the person skilled in the art and include,e.g., Northern Blot.

In the context of the present invention the term “recombinant” meansthat the microorganism is genetically modified so as to contain anucleic acid molecule encoding an enzyme as defined above as compared toa wild-type or non-modified microorganism. A nucleic acid moleculeencoding an enzyme as defined above can be used alone or as part of avector.

The nucleic acid molecules can further comprise expression controlsequences operably linked to the polynucleotide comprised in the nucleicacid molecule. The term “operatively linked” or “operably linked”, asused throughout the present description, refers to a linkage between oneor more expression control sequences and the coding region in thepolynucleotide to be expressed in such a way that expression is achievedunder conditions compatible with the expression control sequence.

Expression comprises transcription of the heterologous DNA sequence,preferably into a translatable mRNA. Regulatory elements ensuringexpression in fungi as well as in bacteria, are well known to thoseskilled in the art. They encompass promoters, enhancers, terminationsignals, targeting signals and the like. Examples are given furtherbelow in connection with explanations concerning vectors.

Promoters for use in connection with the nucleic acid molecule may behomologous or heterologous with regard to its origin and/or with regardto the gene to be expressed. Suitable promoters are for instancepromoters which lend themselves to constitutive expression. However,promoters which are only activated at a point in time determined byexternal influences can also be used. Artificial and/or chemicallyinducible promoters may be used in this context.

The vectors can further comprise expression control sequences operablylinked to said polynucleotides contained in the vectors. Theseexpression control sequences may be suited to ensure transcription andsynthesis of a translatable RNA in bacteria or fungi.

In addition, it is possible to insert different mutations into thepolynucleotides by methods usual in molecular biology (see for instanceSambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSHPress, Cold Spring Harbor, N.Y., USA), leading to the synthesis ofpolypeptides possibly having modified biological properties. Theintroduction of point mutations is conceivable at positions at which amodification of the amino acid sequence for instance influences thebiological activity or the regulation of the polypeptide.

Moreover, mutants possessing a modified substrate or product specificitycan be prepared. Preferably, such mutants show an increased activity.Alternatively, mutants can be prepared the catalytic activity of whichis abolished without losing substrate binding activity.

Furthermore, the introduction of mutations into the polynucleotidesencoding an enzyme as defined above allows the gene expression rateand/or the activity of the enzymes encoded by said polynucleotides to bereduced or increased.

For genetically modifying bacteria or fungi, the polynucleotidesencoding an enzyme as defined above or parts of these molecules can beintroduced into plasmids which permit mutagenesis or sequencemodification by recombination of DNA sequences. Standard methods (seeSambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSHPress, Cold Spring Harbor, N.Y., USA) allow base exchanges to beperformed or natural or synthetic sequences to be added. DNA fragmentscan be connected to each other by applying adapters and linkers to thefragments. Moreover, engineering measures which provide suitablerestriction sites or remove surplus DNA or restriction sites can beused. In those cases, in which insertions, deletions or substitutionsare possible, in vitro mutagenesis, “primer repair”, restriction orligation can be used. In general, a sequence analysis, restrictionanalysis and other methods of biochemistry and molecular biology arecarried out as analysis methods.

Thus, in accordance with the present invention a recombinantmicroorganism can be produced by genetically modifying fungi or bacteriacomprising introducing the above-described polynucleotides, nucleic acidmolecules or vectors into a fungus or bacterium.

The polynucleotide encoding the respective enzyme is expressed so as tolead to the production of a polypeptide having any of the activitiesdescribed above. An overview of different expression systems is forinstance contained in Methods in Enzymology 153 (1987), 385-516, inBitter et al. (Methods in Enzymology 153 (1987), 516-544) and in Sawerset al. (Applied Microbiology and Biotechnology 46 (1996), 1-9),Billman-Jacobe (Current Opinion in Biotechnology 7 (1996), 500-4),Hockney (Trends in Biotechnology 12 (1994), 456-463), Griffiths et al.,(Methods in Molecular Biology 75 (1997), 427-440). An overview of yeastexpression systems is for instance given by Hensing et al. (Antonie vanLeuwenhoek 67 (1995), 261-279), Bussineau et al. (Developments inBiological Standardization 83 (1994), 13-19), Gellissen et al. (Antonievan Leuwenhoek 62 (1992), 79-93, Fleer (Current Opinion in Biotechnology3 (1992), 486-496), Vedvick (Current Opinion in Biotechnology 2 (1991),742-745) and Buckholz (Bio/Technology 9 (1991), 1067-1072).

Expression vectors have been widely described in the literature. As arule, they contain not only a selection marker gene and areplication-origin ensuring replication in the host selected, but also abacterial or viral promoter, and in most cases a termination signal fortranscription. Between the promoter and the termination signal there isin general at least one restriction site or a polylinker which enablesthe insertion of a coding DNA sequence. The DNA sequence naturallycontrolling the transcription of the corresponding gene can be used asthe promoter sequence, if it is active in the selected host organism.However, this sequence can also be exchanged for other promotersequences. It is possible to use promoters ensuring constitutiveexpression of the gene and inducible promoters which permit a deliberatecontrol of the expression of the gene. Bacterial and viral promotersequences possessing these properties are described in detail in theliterature. Regulatory sequences for the expression in microorganisms(for instance E. coli, S. cerevisiae) are sufficiently described in theliterature. Promoters permitting a particularly high expression of adownstream sequence are for instance the T7 promoter (Studier et al.,Methods in Enzymology 185 (1990), 60-89), lacUV5, trp, trp-lacUV5(DeBoer et al., in Rodriguez and Chamberlin (Eds), Promoters, Structureand Function; Praeger, New York, (1982), 462-481; DeBoer et al., Proc.Natl. Acad. Sci. USA (1983), 21-25), Ip1, rac (Boros et al., Gene 42(1986), 97-100). Inducible promoters are preferably used for thesynthesis of polypeptides. These promoters often lead to higherpolypeptide yields than do constitutive promoters. In order to obtain anoptimum amount of polypeptide, a two-stage process is often used. First,the host cells are cultured under optimum conditions up to a relativelyhigh cell density. In the second step, transcription is induceddepending on the type of promoter used. In this regard, a tac promoteris particularly suitable which can be induced by lactose or IPTG(=isopropyl-B-D-thiogalactopyranoside) (deBoer et al., Proc. Natl. Acad.Sci. USA 80 (1983), 21-25). Termination signals for transcription arealso described in the literature.

The transformation of the host cell with a polynucleotide or vector asdescribed above can be carried out by standard methods, as for instancedescribed in Sambrook and Russell (2001), Molecular Cloning: ALaboratory Manual, CSH Press, Cold Spring Harbor, N.Y., USA; Methods inYeast Genetics, A Laboratory Course Manual, Cold Spring HarborLaboratory Press, 1990. The host cell is cultured in nutrient mediameeting the requirements of the particular host cell used, in particularin respect of the pH value, temperature, salt concentration, aeration,antibiotics, vitamins, trace elements etc.

Recombinant Organisms or Microorganisms Expressing Enzymes of Step I andStep II, and Optionally Further Expressing Enzymes of Step III, Step IVand Step V as Well as Optionally Further Expressing Enzymes of StepsXIII, XIV and XV

The present invention also relates to a recombinant organism ormicroorganism which expresses (i) an enzyme capable of enzymaticallyconverting 3-methylcrotonic acid into isobutene (step I as shown in FIG.1); and (ii) an enzyme capable of enzymatically converting3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid (step II as shownin FIG. 1).

In a preferred embodiment, the enzyme capable of converting3-methylcrotonic acid into isobutene is a 3-methylcrotonic aciddecarboxylase as defined herein above. More preferably, the3-methylcrotonic acid decarboxylase is

-   -   (i) an FMN-dependent decarboxylase associated with an FMN prenyl        transferase; or    -   (ii) an aconitate decarboxylase (EC 4.1.1.6); or    -   (iii) a methylcrotonyl-CoA carboxylase (EC 6.4.1.4); or    -   (iv) a geranoyl-CoA carboxylase (EC 6.4.1.5); or    -   (v) a protocatechuate (PCA) decarboxylase (EC 4.1.1.63) as        defined herein above.

In another preferred embodiment, this recombinant organism ormicroorganism is a recombinant organism or microorganism, wherein the3-methylcrotonic acid decarboxylase is selected from the groupconsisting of: 6-methylsalicylate decarboxylase (EC 4.1.1.52),2-oxo-3-hexenedioate decarboxylase (EC 4.1.1.77) and5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylase (EC 4.1.1.68).

As regards the 3-methylcrotonic acid decarboxylase, the FMN-dependentdecarboxylase, the associated FMN prenyl transferase, the aconitatedecarboxylase (EC 4.1.1.6), the methylcrotonyl-CoA carboxylase (EC6.4.1.4), and the geranoyl-CoA carboxylase (EC 6.4.1.5) as well aspreferred embodiments of said 3-methylcrotonic acid decarboxylase, saidprotocatechuate (PCA) decarboxylase (EC 4.1.1.63), said FMN-dependentdecarboxylase, said associated FMN prenyl transferase, said aconitatedecarboxylase (EC 4.1.1.6), said methylcrotonyl-CoA carboxylase (EC6.4.1.4) and said geranoyl-CoA carboxylase (EC 6.4.1.5), as well as said6-methylsalicylate decarboxylase (EC 4.1.1.52), 2-oxo-3-hexenedioatedecarboxylase (EC 4.1.1.77) and 5-oxopent-3-ene-1,2,5-tricarboxylatedecarboxylase (EC 4.1.1.68), the same applies to the recombinantorganism or microorganism as has been set forth above for the methodsaccording to the present invention.

In a preferred embodiment, the recombinant organism or microorganismwhich expresses (i) an enzyme capable of enzymatically converting3-methylcrotonic acid into isobutene (step I as shown in FIG. 1); and(ii) an enzyme capable of enzymatically converting 3-hydroxyisovalerate(HIV) into 3-methylcrotonic acid (step II as shown in FIG. 1) is arecombinant organism or microorganism, wherein the enzyme capable ofenzymatically converting 3-hydroxyisovalerate (HIV) into3-methylcrotonic acid is a hydro-lyase (EC 4.2.-.-) as defined hereinabove, preferably an aconitase (EC 4.2.1.3), a fumarase (EC 4.2.1.2) oran enoyl-CoA hydratase/dehydratease (EC 4.2.1.17) as defined hereinabove.

As regards the hydro-lyase (EC 4.2.-.-), the aconitase (EC 4.2.1.3), thefumarase (EC 4.2.1.2) and the enoyl-CoA hydratase/dehydratease (EC4.2.1.17) as well as the preferred embodiments of said hydro-lyase (EC4.2.-.-), said aconitase (EC 4.2.1.3), said fumarase (EC 4.2.1.2) andsaid enoyl-CoA hydratase/dehydratease (EC 4.2.1.17) the same applies tothe recombinant organism or microorganism as has been set forth abovefor the methods according to the present invention.

In a further aspect, the above recombinant organism or microorganism isan organism or microorganism which further expresses an enzyme capableof enzymatically condensing acetone and acetyl-CoA into3-hydroxyisovalerate (HIV) (step III as shown in FIG. 1). In a preferredembodiment, the enzyme capable of enzymatically condensing acetone andacetyl-CoA into 3-hydroxyisovalerate (HIV) is a HMG CoA synthase (EC2.3.3.10) or a PksG protein or an enzyme with the activity of a C-C bondcleavage/condensation lyase, such as a HMG CoA lyase (EC 4.1.3.4) asdefined herein above.

As regards the HMG CoA synthase (EC 2.3.3.10), the PksG protein, theenzyme with the activity of a C-C bond cleavage/condensation lyase andthe HMG CoA lyase (EC 4.1.3.4) as well as the preferred embodiments ofsaid enzymes the same applies to the recombinant organism ormicroorganism as has been set forth above for the methods according tothe present invention.

In a further aspect, the above recombinant organism or microorganism isan organism or microorganism which further expresses an enzyme capableof enzymatically converting acetoacetate into acetone (step IV as shownin FIG. 1), preferably an acetoacetate decarboxylase (EC 4.1.1.4) asdescribed herein above.

As regards said enzyme capable of enzymatically converting acetoacetateinto acetone and said acetoacetate decarboxylase (EC 4.1.1.4) as well aspreferred embodiments of said enzymes, the same applies to therecombinant organism or microorganism as has been set forth above forthe methods according to the present invention.

In a further aspect, the above recombinant organism or microorganism isan organism or microorganism which further expresses an enzyme capableof converting acetoacetyl-CoA into acetoacetate (step Va or Vb as shownin FIG. 1), preferably

-   -   (i) an acetoacetyl-CoA hydrolase (EC 3.1.2.11); or    -   (ii) an enzyme which is capable of transferring the CoA group of        acetoacetyl-CoA on acetate

as described herein above.

In a preferred embodiment, the enzyme capable of transferring the CoAgroup of acetoacetyl-CoA on acetate is a CoA transferase (EC 2.8.3.-),preferably an acetate CoA transferase (EC 2.8.3.8) as described hereinabove.

As regards said enzyme which is capable of converting acetoacetyl-CoAinto acetoacetate, said acetoacetyl-CoA hydrolase (EC 3.1.2.11), saidenzyme which is capable of transferring the CoA group ofacetoacetyl-CoA, the CoA transferase (EC 2.8.3.-) and said acetate CoAtransferase (EC 2.8.3.8) as well as the preferred embodiments of saidenzymes, the same applies to the recombinant organism or microorganismas has been set forth above for the methods according to the presentinvention.

In a further aspect, the above recombinant organism or microorganism isan organism or microorganism which further expresses an enzyme capableof enzymatically converting acetyl-CoA into acetoacetyl-CoA comprising

-   -   (a) (i) an enzyme capable of converting acetyl-CoA into        malonyl-CoA (step XIV as shown in FIG. 1); and        -   (ii) an enzyme capable of condensing malonyl-CoA and            acetyl-CoA into acetoacetyl-CoA (step XV as shown in FIG.            1); or    -   (b) an enzyme capable of directly condensing two molecules of        acetyl-CoA into acetoacetyl-CoA (step XIII as shown in FIG. 1).

In a preferred embodiment, the enzyme capable of converting acetyl-CoAinto malonyl-CoA is an acetyl-CoA carboxylase (EC 6.4.1.2) as describedherein above.

In another preferred embodiment, the enzyme capable of condensingmalonyl-CoA and acetyl-CoA into acetoacetyl-CoA is an acetoacetyl-CoAsynthetase (EC 2.3.1.194) as described herein above.

In a preferred embodiment, the enzyme capable of directly condensing twomolecules of acetyl-CoA into acetoacetyl-CoA is an acetyl-CoAC-acetyltransferase (EC 2.3.1.9) as described herein above.

As regards the enzyme which is capable of converting acetyl-CoA intomalonyl-CoA, the enzyme capable of condensing malonyl-CoA and acetyl-CoAinto acetoacetyl-CoA, the acetyl-CoA carboxylase (EC 6.4.1.2), theacetoacetyl-CoA synthetase (EC 2.3.1.194), the enzyme capable ofdirectly condensing two molecules of acetyl-CoA into acetoacetyl-CoA andthe acetyl-CoA C-acetyltransferase (EC 2.3.1.9) as well as the preferredembodiments of said enzymes, the same applies to the recombinantorganism or microorganism as has been set forth above for the methodsaccording to the present invention.

Recombinant Organisms or Microorganisms Expressing Enzymes of Step I andStep VI, and Optionally Further Expressing Enzymes of Step VII, StepVIII and Step IX as Well as Optionally Further Expressing Enzymes ofSteps XIII, XIV and XV

The present invention also relates to a recombinant organism ormicroorganism which expresses (i) an enzyme capable of enzymaticallyconverting 3-methylcrotonic acid into isobutene (step I as shown in FIG.1); and (ii) an enzyme capable of enzymatically converting3-methylcrotonyl-CoA into 3-methylcrotonic acid (step VIa, VIb or VIc asshown in FIG. 1).

In a preferred embodiment, the enzyme capable of converting3-methylcrotonic acid into isobutene is a 3-methylcrotonic aciddecarboxylase, preferably

-   -   (i) an FMN-dependent decarboxylase associated with an FMN prenyl        transferase; or    -   (ii) an aconitate decarboxylase (EC 4.1.1.6); or    -   (iii) a methylcrotonyl-CoA carboxylase (EC 6.4.1.4); or    -   (iv) a geranoyl-CoA carboxylase (EC 6.4.1.5); or    -   (v) a protocatechuate (PCA) decarboxylase (EC 4.1.1.63) as        defined herein above.

As regards the 3-methylcrotonic acid decarboxylase, the FMN-dependentdecarboxylase, the associated FMN prenyl transferase, the aconitatedecarboxylase (EC 4.1.1.6), the methylcrotonyl-CoA carboxylase (EC6.4.1.4), the (v) protocatechuate (PCA) decarboxylase (EC 4.1.1.63) andthe geranoyl-CoA carboxylase (EC 6.4.1.5) as well as preferredembodiments of said enzymes, the same applies to the recombinantorganism or microorganism as has been set forth above for the methodsaccording to the present invention.

In a preferred embodiment, the enzyme capable of enzymaticallyconverting 3-methylcrotonyl-CoA into 3-methylcrotonic acid is

-   -   (a) an enzyme capable of directly converting        3-methylcrotonyl-CoA into 3-methylcrotonic acid wherein said        enzyme capable of directly converting 3-methylcrotonyl-CoA into        3-methylcrotonic acid is a CoA transferase (EC 2.8.3.-),        preferably a propionate:acetate-CoA transferase (EC 2.8.3.1), an        acetate CoA-transferase (EC 2.8.3.8) or a succinyl-CoA:acetate        CoA-transferase (EC 2.8.3.18) (step VIa as shown in FIG. 1) as        described herein above; or    -   (b) an enzyme capable of directly converting        3-methylcrotonyl-CoA into 3-methylcrotonic acid wherein said        enzyme capable of directly converting 3-methylcrotonyl-CoA into        3-methylcrotonic acid is a thioester hydrolase (EC 3.1.2.-),        preferably acetyl-CoA hydrolase (EC 3.1.2.1), an ADP-dependent        short-chain-acyl-CoA hydrolase (EC 3.1.2.18) or an acyl-CoA        hydrolase (EC 3.1.2.20) (step VIb as shown in FIG. 1) as        described herein above.

In another preferred embodiment, the recombinant organism ormicroorganism is a recombinant organism or microorganism which expressesthe following two enzymes, namely

-   -   (c) (i) an enzyme capable of enzymatically converting        3-methylcrotonyl-CoA into 3-methylcrotonyl phosphate as        described herein above; and        -   (ii) an enzyme capable of converting 3-methylcrotonyl            phosphate into 3-methylcrotonic acid (step VIc as shown in            FIG. 1) as described herein above.

In a preferred embodiment, the enzyme capable of converting3-methylcrotonyl-CoA into 3-methylcrotonyl phosphate is a phosphatebutyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC2.3.1.8) and the enzyme capable of converting 3-methylcrotonyl phosphateinto 3-methylcrotonic acid is a phosphotransferase with a carboxy groupas acceptor (EC 2.7.2.-), preferably a propionate kinase (EC 2.7.2.15),an acetate kinase (EC 2.7.2.1), a butyrate kinase (EC 2.7.2.7) or abranched-chain-fatty-acid kinase (EC 2.7.2.14) as described hereinabove.

As regards the above-mentioned enzymes, the same applies to therecombinant organism or microorganism as has been set forth above forthe methods according to the present invention.

In a further aspect, the above recombinant organism or microorganism isan organism or microorganism which further expresses an enzyme capableof enzymatically converting 3-methylglutaconyl-CoA into3-methylcrotonyl-CoA (step VII as shown in FIG. 1), preferably (i) amethylcrotonyl-CoA carboxylase (EC 6.4.1.4); or (ii) a geranoyl-CoAcarboxylase (EC 6.4.1.5) as described herein above.

As regards said enzymes as well as preferred embodiments of saidenzymes, the same applies to the recombinant organism or microorganismas has been set forth above for the methods according to the presentinvention.

In a further aspect, the above recombinant organism or microorganism isan organism or microorganism which further expresses an enzyme capableof enzymatically converting 3-hydroxy-3-methylglutaryl-CoA into3-methylglutaconyl-CoA (step VIII as shown in FIG. 1), preferably a3-methylglutaconyl-coenzyme A hydratase (EC 4.2.1.18), a3-hydroxyacyl-CoA dehydratase (EC 4.2.1.-) or an enoyl-CoA hydratase (EC4.2.1.-).

As regards said enzyme as well as preferred embodiments of said enzymes,the same applies to the recombinant organism or microorganism as hasbeen set forth above for the methods according to the present invention.

In a further aspect, the above recombinant organism or microorganism isan organism or microorganism which further expresses an enzyme capableof enzymatically condensing acetoacetyl-CoA and acetyl-CoA into3-hydroxy-3-methylglutaryl-CoA (step IX as shown in FIG. 1), preferablya 3-hydroxy-3-methylglutaryl-CoA synthase.

As regards said enzyme as well as preferred embodiments of said enzyme,the same applies to the recombinant organism or microorganism as hasbeen set forth above for the methods according to the present invention.

In a further aspect, the above recombinant organism or microorganismwhich expresses (i) an enzyme capable of enzymatically converting3-methylcrotonic acid into isobutene (step I as shown in FIG. 1); and(ii) an enzyme capable of enzymatically converting 3-methylcrotonyl-CoAinto 3-methylcrotonic acid (step VIa, VIb or VIc as shown in FIG. 1)(and optionally further expressing an enzyme capable of enzymaticallyconverting 3-methylglutaconyl-CoA into 3-methylcrotonyl-CoA andoptionally further expressing an enzyme capable of enzymaticallyconverting 3-hydroxy3-methylglutaryl-CoA into 3-methylgutaconyl-CoA andoptionally further expressing an enzyme capable of enzymaticallycondensing acetoacetyl-CoA and acetyl-CoA into3-hydroxy-3-methylglutaryl-CoA) is preferably an organism ormicroorganism which further expresses an enzyme capable of enzymaticallyconverting acetyl-CoA into acetoacetyl-CoA comprising

an enzyme capable of directly condensing two molecules of acetyl-CoAinto acetoacetyl-CoA (step XIII as shown in FIG. 1).

In another preferred embodiment, the recombinant organism ormicroorganism is a recombinant organism or microorganism which expressesthe following two enzymes, namely

-   -   (i) an enzyme capable of converting acetyl-CoA into malonyl-CoA        (step XIV as shown in FIG. 1); and    -   (ii) an enzyme capable of condensing malonyl-CoA and acetyl-CoA        into acetoacetyl-CoA (step XV as shown in FIG. 1).

In a preferred embodiment, the enzyme capable of converting acetyl-CoAinto malonyl-CoA is an acetyl-CoA carboxylase (EC 6.4.1.2) as describedherein above.

In another preferred embodiment, the enzyme capable of condensingmalonyl-CoA and acetyl-CoA into acetoacetyl-CoA is an acetoacetyl-CoAsynthetase (EC 2.3.1.194) as described herein above.

In a preferred embodiment, the enzyme capable of directly condensing twomolecules of acetyl-CoA into acetoacetyl-CoA is an acetyl-CoAC-acetyltransferase (EC 2.3.1.9) as described herein above.

As regards the above-mentioned enzymes as well as the preferredembodiments of said enzymes, the same applies to the recombinantorganism or microorganism as has been set forth above for the methodsaccording to the present invention.

Recombinant Organisms or Microorganisms of the Alternative Route for theEnzymatic Conversion from Acetyl-CoA into Isobutene via3-Methyl-3-Butenovl-CoA and 3-Methyl-3-Butenoic Acid: RecombinantOrganisms or Microorganisms Expressing Enzymes of Step XVI and StepXVII, and Optionally Further Expressing Enzymes of Step XVIII, Step VIIIand Step IX as Well as Optionally Further Expressing Enzymes of StepsXIII, XIV and XV

As mentioned above, in an alternative to the above first route for theproduction of isobutene via 3-methylcrotonic acid, the present inventionalso relates to a method for the production of isobutene via analternative route wherein isobutene is produced by the enzymaticconversion of 3-methyl-3-butenoic acid into isobutene. In the following,the recombinant organisms or microorganisms of this alternative routefor the enzymatic conversion from acetyl-CoA into isobutene via3-methyl-3-butenoyl-CoA and 3-methyl-3-butenoic acid are described.

The present invention also relates to a recombinant organism ormicroorganism which expresses (i) an enzyme capable of enzymaticallyconverting 3-methyl-3-butenoic acid into isobutene (step XVI as shown inFIG. 1) and (ii) an enzyme capable of enzymatically converting3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoic acid (step XVII asshown in FIG. 1).

In a preferred embodiment, the enzyme capable of enzymaticallyconverting 3-methyl-3-butenoic acid into isobutene is an3-methyl-3-butenoic acid decarboxylase as described herein above, morepreferably

-   -   (i) an FMN-dependent decarboxylase associated with an FMN prenyl        transferase; or    -   (ii) an aconitate decarboxylase (EC 4.1.1.6); or    -   (iii) a methylcrotonyl-CoA carboxylase (EC 6.4.1.4); or    -   (iv) a geranoyl-CoA carboxylase (EC 6.4.1.5); or    -   (v) a protocatechuate (PCA) decarboxylase (EC 4.1.1.63) as        described herein above.

In another preferred embodiment, the 3-methyl-3-butenoic aciddecarboxylase is selected from the group consisting of6-methylsalicylate decarboxylase (EC 4.1.1.52), 2-oxo-3-hexenedioatedecarboxylase (EC 4.1.1.77) and 5-oxopent-3-ene-1,2,5-tricarboxylatedecarboxylase (EC 4.1.1.68) as described herein above.

As regards the above-mentioned enzymes as well as preferred embodimentsof said enzymes, the same applies to the recombinant organism ormicroorganism as has been set forth above for the methods according tothe present invention.

In a preferred embodiment, the enzyme capable of enzymaticallyconverting 3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoic acid is

-   -   (a) an enzyme capable of directely converting        3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoic acid, wherein        said enzyme capable of directely converting        3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoic acid is a CoA        transferase (EC 2.8.3.-), preferably a propionate:acetate-CoA        transferase (EC 2.8.3.1), an acetate CoA-transferase (EC        2.8.3.8) or a succinyl-CoA:acetate CoA-transferase (EC 2.8.3.18)        (step XVIIa as shown in FIG. 1) as described herein above.

In another preferred embodiment, the recombinant organism ormicroorganism is a recombinant organism or microorganism which expressesthe following two enzymes, namely

-   -   (b) an enzyme capable of directely converting        3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoic acid, wherein        said enzyme capable of directely converting        3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoic acid is a        thioester hydrolase (EC 3.1.2.-), preferably acetyl-CoA        hydrolase (EC 3.1.2.1), an ADP-dependent short-chain-acyl-CoA        hydrolase (EC 3.1.2.18) or an acyl-CoA hydrolase (EC 3.1.2.20)        (step XVIIb as shown in FIG. 1) as described herein above; or    -   (c) (i) an enzyme capable of enzymatically converting        3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoyl phosphate; and        -   (ii) an enzyme capable of enzymatically converting            3-methyl-3-butenoyl phosphate into said 3-methyl-3-butenoic            acid(step XVIII as shown in FIG. 1) as described herein            above.

In a preferred embodiment, the enzyme capable of enzymaticallyconverting said 3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoylphosphate is a phosphate butyryltransferase (EC 2.3.1.19) or a phosphateacetyltransferase (EC 2.3.1.8) and the enzyme capable of enzymaticallyconverting 3-methyl-3-butenoyl phosphate into 3-methyl-3-butenoic acidis a phosphotransferase with a carboxy group as acceptor (EC 2.7.2.-),preferably a propionate kinase (EC 2.7.2.15), an acetate kinase (EC2.7.2.1), a butyrate kinase (EC 2.7.2.7) or a branched-chain-fatty-acidkinase (EC 2.7.2.14) as described herein above.

As regards the above-mentioned enzymes as well as preferred embodimentsof said enzymes, the same applies to the recombinant organism ormicroorganism as has been set forth above for the methods according tothe present invention.

In a further aspect, the above recombinant organism or microorganism isan organism or microorganism which further expresses an enzyme capableof enzymatically converting 3-methylglutaconyl-CoA into3-methyl-3-butenoyl-CoA (step XVIII as shown in FIG. 1), preferably

-   -   (a) (i) a methylcrotonyl-CoA carboxylase (EC 6.4.1.4); or (ii) a        geranoyl-CoA carboxylase (EC 6.4.1.5), or    -   (b) an N-terminal domain of CurF from Lynbya majuscula        multifunctional protein or a 3-methylglutaconyl-CoA        decarboxylase, preferably a 3-methylglutaconyl-CoA decarboxylase        of Myxococcus xanthus encoded by the liuB gene; or    -   (c) an enzyme of the 4-oxalocrotonate decarboxylase family, as        described herein above.

As regards the above-mentioned enzymes as well as preferred embodimentsof said enzymes, the same applies to the recombinant organism ormicroorganism as has been set forth above for the methods according tothe present invention.

In a further aspect, the above recombinant organism or microorganism isan organism or microorganism which further expresses an enzyme capableof enzymatically converting 3-hydroxy-3-methylglutaryl-CoA into3-methylglutaconyl-CoA (step VIII as shown in FIG. 1),preferably a3-methylglutaconyl-coenzyme A hydratase (EC 4.2.1.18), a3-hydroxyacyl-CoA dehydratase (EC 4.2.1.-) or an enoyl-CoA hydratase (EC4.2.1.-).

As regards the above-mentioned enzyme as well as preferred embodimentsof said enzyme, the same applies to the recombinant organism ormicroorganism as has been set forth above for the methods according tothe present invention.

In a further aspect, the above recombinant organism or microorganism isan organism or microorganism which further expresses an enzyme capableof enzymatically condensing acetoacetyl-CoA and acetyl-CoA into3-hydroxy-3-methylglutaryl-CoA (step IX as shown in FIG. 1).

In a preferred embodiment, the enzyme capable of enzymaticallycondensing acetoacetyl-CoA and acetyl-CoA into3-hydroxy-3-methylglutaryl-CoA is a 3-hydroxy-3-methylglutaryl-CoAsynthase.

As regards the afore-mentioned enzyme as well as preferred embodimentsof said enzyme, the same applies to the recombinant organism ormicroorganism as has been set forth above for the methods according tothe present invention.

In a further aspect, the above recombinant organism or microorganism isan organism or microorganism which further expresses an enzyme orseveral enzymes capable of enzymatically converting acetyl-CoA intoacetoacetyl-CoA.

In one preferred embodiment, the recombinant organism or microorganismexpresses a combination of enzymes, namely

-   -   (i) an enzyme capable of converting acetyl-CoA into malonyl-CoA        (step XIV as shown in FIG. 1); and    -   (ii) an enzyme capable of condensing malonyl-CoA and acetyl-CoA        into acetoacetyl-CoA (step XV as shown in FIG. 1).

In an alternative embodiment, the recombinant organism or microorganismexpresses an enzyme capable of directly condensing two molecules ofacetyl-CoA into acetoacetyl-CoA (step XIII as shown in FIG. 1).

As regards the first above-mentioned embodiment, the enzyme capable ofconverting acetyl-CoA into malonyl-CoA is preferably an acetyl-CoAcarboxylase (EC 6.4.1.2) as described herein above.

Moreover, the enzyme capable of condensing malonyl-CoA and acetyl-CoAinto acetoacetyl-CoA is an acetoacetyl-CoA synthetase (EC 2.3.1.194) asdescribed herein above.

As regards the second above-mentioned embodiment, the enzyme capable ofdirectly condensing two molecules of acetyl-CoA into acetoacetyl-CoA ispreferably an acetyl-CoA C-acetyltransferase (EC 2.3.1.9) as describedherein above.

As regards the above-mentioned enzymes as well as the preferredembodiments of said enzymes, the same applies to the recombinantorganism or microorganism as has been set forth above for the methodsaccording to the present invention.

Recombinant Organisms or Microorganisms Expressing Enzymes of theAdditional/Supplemental Pathways of Steps Xa, Xb, XI and XII

As mentioned above, the above-described methods of the present inventionfor producing isobutene from acetyl-CoA may be supplemented by one ormore of the reactions as shown in step Xa, step Xb, step XI and step XIIof FIG. 18 and as described in detail herein above.

Thus, in a further aspect, the present invention relates to any of theabove-described recombinant organism or microorganism wherein theorganism or microorganism which additionally further expresses

-   -   a) an enzyme capable of enzymatically converting        3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid with a        concomitant transfer of CoA from 3-methylcrotonyl-CoA on        3-hydroxyisovalerate (HIV) to result in 3-hydroxyisovaleryl-CoA        (step Xa as schematically shown in FIG. 19); and/or    -   b) an enzyme capable of enzymatically converting        3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA (step Xb        as schematically shown in FIG. 20); and/or    -   c) an enzyme capable of enzymatically converting        3-hydroxyisovaleryl-CoA into 3-methylcrotonyl-CoA (step XI as        schematically shown in FIG. 21); and/or    -   d) an enzyme capable of enzymatically converting        3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA (step        XII as schematically shown in FIG. 22) as described herein        above.

As regards the above-mentioned enzymes as well as preferred embodimentsof said enzymes, the same applies to the recombinant organism ormicroorganism as has been set forth above for the methods according tothe present invention.

The above microorganism is preferably a bacterium, a yeast or a fungus.In another preferred embodiment, the organism is a plant or a non-humananimal. As regards other preferred embodiments of the bacterium,recombinant organism or microorganism, the same applies as has been setforth above in connection with the methods according to the presentinvention.

The present invention also relates to the use of any of theabove-described recombinant organisms or microorganisms for theproduction of isobutene. Thus, the present invention furthermore relatesto the use of a recombinant organism or microorganism for the productionof isobutene, wherein said recombinant organism or microorganismexpresses (i) an enzyme capable of enzymatically converting3-methylcrotonic acid into isobutene (step I as shown in FIG. 1); and(ii) an enzyme capable of enzymatically converting 3-hydroxyisovalerate(HIV) into 3-methylcrotonic acid (step II as shown in FIG. 1).

In another preferred embodiment, the present invention relates to theuse of a recombinant organism or microorganism for the production ofisobutene, wherein said recombinant organism or microorganism expresses(i) an enzyme capable of enzymatically converting 3-methylcrotonic acidinto isobutene (step I as shown in FIG. 1); and (ii) an enzyme capableof enzymatically converting 3-hydroxyisovalerate (HIV) into3-methylcrotonic acid (step II as shown in FIG. 1) which furtherexpresses an enzyme capable of enzymatically condensing acetone andacetyl-CoA into 3-hydroxyisovalerate (HIV) (step III as shown in FIG.1).

In another preferred embodiment, the present invention relates to theuse of a recombinant organism or microorganism for the production ofisobutene, wherein said recombinant organism or microorganism expresses(i) an enzyme capable of enzymatically converting 3-methylcrotonic acidinto isobutene (step I as shown in FIG. 1); and (ii) an enzyme capableof enzymatically converting 3-hydroxyisovalerate (HIV) into3-methylcrotonic acid (step II as shown in FIG. 1), which furtherexpresses an enzyme capable of enzymatically condensing acetone andacetyl-CoA into 3-hydroxyisovalerate (HIV) (step III as shown in FIG. 1)and which further expresses an enzyme capable of enzymaticallyconverting acetoacetate into acetone (step IV as shown in FIG. 1).

In another preferred embodiment, the present invention relates to theuse of a recombinant organism or microorganism for the production ofisobutene, wherein said recombinant organism or microorganism expresses(i) an enzyme capable of enzymatically converting 3-methylcrotonic acidinto isobutene (step I as shown in FIG. 1); and (ii) an enzyme capableof enzymatically converting 3-hydroxyisovalerate (HIV) into3-methylcrotonic acid (step II as shown in FIG. 1), which furtherexpresses an enzyme capable of enzymatically condensing acetone andacetyl-CoA into 3-hydroxyisovalerate (HIV) (step III as shown in FIG.1), which further expresses an enzyme capable of enzymaticallyconverting acetoacetate into acetone (step IV as shown in FIG. 1) andwhich further expresses an enzyme capable of converting acetoacetyl-CoAinto acetoacetate (step Va or Vb as shown in FIG. 1).

In another preferred embodiment, the present invention relates to theuse of a recombinant organism or microorganism for the production ofisobutene, wherein said recombinant organism or microorganism expresses(i) an enzyme capable of enzymatically converting 3-methylcrotonic acidinto isobutene (step I as shown in FIG. 1); and (ii) an enzyme capableof enzymatically converting 3-hydroxyisovalerate (HIV) into3-methylcrotonic acid (step II as shown in FIG. 1), which furtherexpresses an enzyme capable of enzymatically condensing acetone andacetyl-CoA into 3-hydroxyisovalerate (HIV) (step III as shown in FIG.1), which further expresses an enzyme capable of enzymaticallyconverting acetoacetate into acetone (step IV as shown in FIG. 1), whichfurther expresses an enzyme capable of converting acetoacetyl-CoA intoacetoacetate (step Va or Vb as shown in FIG. 1) and which furtherexpresses an enzyme capable of enzymatically converting acetyl-CoA intoacetoacetyl-CoA comprising (a) (i) an enzyme capable of convertingacetyl-CoA into malonyl-CoA (step XIV as shown in FIG. 1); and (ii) anenzyme capable of condensing malonyl-CoA and acetyl-CoA intoacetoacetyl-CoA (step XV as shown in FIG. 1); or (b) an enzyme capableof directly condensing two molecules of acetyl-CoA into acetoacetyl-CoA(step XIII as shown in FIG. 1).

In a more preferred embodiment, the present invention relates to any ofthe above uses of a recombinant organisms or microorganisms for theproduction of isobutene wherein said recombinant organism ormicroorganism expresses an enzyme catalyzing the enzymatic conversion of3-methylcrotonic acid into isobutene.

As regards the above-mentioned enzymes as well as preferred embodimentsof said enzymes, the same applies to the use of the recombinant organismor microorganism for the production of isobutene as has been set forthabove for the methods and recombinant organisms or microorganismsaccording to the present invention.

The present invention furthermore relates to the use of a recombinantorganism or microorganism for the production of isobutene, wherein saidrecombinant organism or microorganism expresses (i) an enzyme capable ofenzymatically converting 3-methylcrotonic acid into isobutene (step I asshown in FIG. 1); and (ii) an enzyme capable of enzymatically converting3-methylcrotonyl-CoA into 3-methylcrotonic acid (step VIa, VIb or VIc asshown in FIG. 1).

In another preferred embodiment, the present invention relates to theuse of a recombinant organism or microorganism for the production ofisobutene, wherein said recombinant organism or microorganism expresses(i) an enzyme capable of enzymatically converting 3-methylcrotonic acidinto isobutene (step I as shown in FIG. 1); and (ii) an enzyme capableof enzymatically converting 3-methylcrotonyl-CoA into 3-methylcrotonicacid (step VIa, VIb or VIc as shown in FIG. 1) and which furtherexpresses an enzyme capable of enzymatically converting3-methylglutaconyl-CoA into 3-methylcrotonyl-CoA (step VII as shown inFIG. 1).

In another preferred embodiment, the present invention relates to theuse of a recombinant organism or microorganism for the production ofisobutene, wherein said recombinant organism or microorganism expresses(i) an enzyme capable of enzymatically converting 3-methylcrotonic acidinto isobutene (step I as shown in FIG. 1); and (ii) an enzyme capableof enzymatically converting 3-methylcrotonyl-CoA into 3-methylcrotonicacid (step VIa, VIb or VIc as shown in FIG. 1), which further expressesan enzyme capable of enzymatically converting 3-methylglutaconyl-CoAinto 3-methylcrotonyl-CoA (step VII as shown in FIG. 1) and whichfurther expresses an enzyme capable of enzymatically converting3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA (step VIII asshown in FIG. 1).

In another preferred embodiment, the present invention relates to theuse of a recombinant organism or microorganism for the production ofisobutene, wherein said recombinant organism or microorganism expresses(i) an enzyme capable of enzymatically converting 3-methylcrotonic acidinto isobutene (step I as shown in FIG. 1); and (ii) an enzyme capableof enzymatically converting 3-methylcrotonyl-CoA into 3-methylcrotonicacid (step VIa, VIb or VIc as shown in FIG. 1), which further expressesan enzyme capable of enzymatically converting 3-methylglutaconyl-CoAinto 3-methylcrotonyl-CoA (step VII as shown in FIG. 1), which furtherexpresses an enzyme capable of enzymatically converting3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA (step VIII asshown in FIG. 1) and which further expresses an enzyme capable ofenzymatically condensing acetoacetyl-CoA and acetyl-CoA into3-hydroxy-3-methylglutaryl-CoA (step IX as shown in FIG. 1).

In another preferred embodiment, the present invention relates to theuse of a recombinant organism or microorganism for the production ofisobutene, wherein said recombinant organism or microorganism expresses(i) an enzyme capable of enzymatically converting 3-methylcrotonic acidinto isobutene (step I as shown in FIG. 1); and (ii) an enzyme capableof enzymatically converting 3-methylcrotonyl-CoA into 3-methylcrotonicacid (step VIa, VIb or VIc as shown in FIG. 1), which further expressesan enzyme capable of enzymatically converting 3-methylglutaconyl-CoAinto 3-methylcrotonyl-CoA (step VII as shown in FIG. 1), which furtherexpresses an enzyme capable of enzymatically converting3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA (step VIII asshown in FIG. 1), which further expresses an enzyme capable ofenzymatically condensing acetoacetyl-CoA and acetyl-CoA into3-hydroxy-3-methylglutaryl-CoA (step IX as shown in FIG. 1) and whichfurther expresses an enzyme capable of enzymatically convertingacetyl-CoA into acetoacetyl-CoA comprising (a) (i) an enzyme capable ofconverting acetyl-CoA into malonyl-CoA (step XIV as shown in FIG. 1);and (ii) an enzyme capable of condensing malonyl-CoA and acetyl-CoA intoacetoacetyl-CoA (step XV as shown in FIG. 1); or (b) an enzyme capableof directly condensing two molecules of acetyl-CoA into acetoacetyl-CoA(step XIII as shown in FIG. 1).

In a more preferred embodiment, the present invention relates to any ofthe above uses of a recombinant organisms or microorganisms for theproduction of isobutene wherein said recombinant organism ormicroorganism expresses an enzyme catalyzing the enzymatic conversion of3-methylcrotonic acid into isobutene.

As regards the above-mentioned enzymes as well as preferred embodimentsof said enzymes, the same applies to the use of the recombinant organismor microorganism for the production of isobutene as has been set forthabove for the mehtods and recombinant organisms or microorganismsaccording to the present invention.

The present invention furthermore relates to the use of a recombinantorganism or microorganism for the production of isobutene, wherein saidrecombinant organism or microorganism expresses (i) an enzyme capable ofenzymatically converting 3-methyl-3-butenoic acid into isobutene (stepXVI as shown in FIG. 1) and (ii) an enzyme capable of enzymaticallyconverting 3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoic acid (stepXVII as shown in FIG. 1).

In another preferred embodiment, the present invention relates to theuse of a recombinant organism or microorganism for the production ofisobutene, wherein said recombinant organism or microorganism expresses(i) an enzyme capable of enzymatically converting 3-methyl-3-butenoicacid into isobutene (step XVI as shown in FIG. 1) and (ii) an enzymecapable of enzymatically converting 3-methyl-3-butenoyl-CoA into3-methyl-3-butenoic acid (step XVII as shown in FIG. 1) and whichfurther expresses an enzyme capable of enzymatically converting3-methylglutaconyl-CoA into 3-methyl-3-butenoyl-CoA (step XVIII as shownin FIG. 1).

In another preferred embodiment, the present invention relates to theuse of a recombinant organism or microorganism for the production ofisobutene, wherein said recombinant organism or microorganism expresses(i) an enzyme capable of enzymatically converting 3-methyl-3-butenoicacid into isobutene (step XVI as shown in FIG. 1) and (ii) an enzymecapable of enzymatically converting 3-methyl-3-butenoyl-CoA into3-methyl-3-butenoic acid (step XVII as shown in FIG. 1), which furtherexpresses an enzyme capable of enzymatically converting3-methylglutaconyl-CoA into 3-methyl-3-butenoyl-CoA (step XVIII as shownin FIG. 1) and which further expresses an enzyme capable ofenzymatically converting 3-hydroxy-3-methylglutaryl-CoA into3-methylglutaconyl-CoA (step VIII as shown in FIG. 1).

In another preferred embodiment, the present invention relates to theuse of a recombinant organism or microorganism for the production ofisobutene, wherein said recombinant organism or microorganism expresses(i) an enzyme capable of enzymatically converting 3-methyl-3-butenoicacid into isobutene (step XVI as shown in FIG. 1) and (ii) an enzymecapable of enzymatically converting 3-methyl-3-butenoyl-CoA into3-methyl-3-butenoic acid (step XVII as shown in FIG. 1), which furtherexpresses an enzyme capable of enzymatically converting3-methylglutaconyl-CoA into 3-methyl-3-butenoyl-CoA (step XVIII as shownin FIG. 1), which further expresses an enzyme capable of enzymaticallyconverting 3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA(step VIII as shown in FIG. 1) and which further expresses an enzymecapable of enzymatically condensing acetoacetyl-CoA and acetyl-CoA into3-hydroxy-3-methylglutaryl-CoA (step IX as shown in FIG. 1).

In another preferred embodiment, the present invention relates to theuse of a recombinant organism or microorganism for the production ofisobutene, wherein said recombinant organism or microorganism expresses(i) an enzyme capable of enzymatically converting 3-methyl-3-butenoicacid into isobutene (step XVI as shown in FIG. 1) and (ii) an enzymecapable of enzymatically converting 3-methyl-3-butenoyl-CoA into3-methyl-3-butenoic acid (step XVII as shown in FIG. 1), which furtherexpresses an enzyme capable of enzymatically converting3-methylglutaconyl-CoA into 3-methyl-3-butenoyl-CoA (step XVIII as shownin FIG. 1), which further expresses an enzyme capable of enzymaticallyconverting 3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA(step VIII as shown in FIG. 1), which further expresses an enzymecapable of enzymatically condensing acetoacetyl-CoA and acetyl-CoA into3-hydroxy-3-methylglutaryl-CoA (step IX as shown in FIG. 1) and whichfurther expresses an enzyme capable of enzymatically convertingacetyl-CoA into acetoacetyl-CoA comprising (a) (i) an enzyme capable ofconverting acetyl-CoA into malonyl-CoA (step XIV as shown in FIG. 1);and (ii) an enzyme capable of condensing malonyl-CoA and acetyl-CoA intoacetoacetyl-CoA (step XV as shown in FIG. 1); or (b) an enzyme capableof directly condensing two molecules of acetyl-CoA into acetoacetyl-CoA(step XIII as shown in FIG. 1).

In a more preferred embodiment, the present invention relates to any ofthe above uses of a recombinant organisms or microorganisms for theproduction of isobutene wherein said recombinant organism ormicroorganism expresses an enzyme catalyzing the enzymatic conversion of3-methyl-3-butenoic acid into isobutene.

As regards the above-mentioned enzymes as well as preferred embodimentsof said enzymes, the same applies to the use of the recombinant organismor microorganism for the production of isobutene as has been set forthabove for the mehtods and recombinant organisms or microorganismsaccording to the present invention.

In a further aspect, the present invention relates to the use of any ofthe above-described recombinant organism or microorganism for theproduction of isobutene, wherein the organism or microorganism is anorganism or microorganism which additionally further expresses

-   -   a) an enzyme capable of enzymatically converting        3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid with a        concomitant transfer of CoA from 3-methylcrotonyl-CoA on        3-hydroxyisovalerate (HIV) to result in 3-hydroxyisovaleryl-CoA        (step Xa as schematically shown in FIG. 19); and/or    -   b) an enzyme capable of enzymatically converting        3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA (step Xb        as schematically shown in FIG. 20); and/or    -   c) an enzyme capable of enzymatically converting        3-hydroxyisovaleryl-CoA into 3-methylcrotonyl-CoA (step XI as        schematically shown in FIG. 21); and/or    -   d) an enzyme capable of enzymatically converting        3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA (step        XII as schematically shown in FIG. 22)

as described herein above.

As regards the abvove-mentioned enzymes as well as preferred embodimentsof said enzymes, the same applies to the recombinant organism ormicroorganism as has been set forth above for the methods according tothe present invention.

The present invention furthermore relates to the use of an enzymecatalyzing the enzymatic conversion of 3-methylcrotonic acid intoisobutene for the production of isobutene from 3-methylcrotonic acid.

The present invention furthermore relates to the use of (i) an enzymecapable of enzymatically converting 3-methylcrotonic acid into isobutene(step I as shown in FIG. 1); and (ii) an enzyme capable of enzymaticallyconverting 3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid (stepII as shown in FIG. 1) for the production of isobutene.

In another preferred embodiment, the present invention relates to theuse (i) an enzyme capable of enzymatically converting 3-methylcrotonicacid into isobutene (step I as shown in FIG. 1); and (ii) an enzymecapable of enzymatically converting 3-hydroxyisovalerate (HIV) into3-methylcrotonic acid (step II as shown in FIG. 1) and an enzyme capableof enzymatically condensing acetone and acetyl-CoA into3-hydroxyisovalerate (HIV) (step III as shown in FIG. 1) for theproduction of isobutene.

In another preferred embodiment, the present invention relates to theuse of (i) an enzyme capable of enzymatically converting3-methylcrotonic acid into isobutene (step I as shown in FIG. 1); and(ii) an enzyme capable of enzymatically converting 3-hydroxyisovalerate(HIV) into 3-methylcrotonic acid (step II as shown in FIG. 1), an enzymecapable of enzymatically condensing acetone and acetyl-CoA into3-hydroxyisovalerate (HIV) (step III as shown in FIG. 1) and an enzymecapable of enzymatically converting acetoacetate into acetone (step IVas shown in FIG. 1) for the production of isobutene.

In another preferred embodiment, the present invention relates to theuse of (i) an enzyme capable of enzymatically converting3-methylcrotonic acid into isobutene (step I as shown in FIG. 1); and(ii) an enzyme capable of enzymatically converting 3-hydroxyisovalerate(HIV) into 3-methylcrotonic acid (step II as shown in FIG. 1); an enzymecapable of enzymatically condensing acetone and acetyl-CoA into3-hydroxyisovalerate (HIV) (step III as shown in FIG. 1), an enzymecapable of enzymatically converting acetoacetate into acetone (step IVas shown in FIG. 1) and an enzyme capable of converting acetoacetyl-CoAinto acetoacetate (step Va or Vb as shown in FIG. 1) for the productionof isobutene.

In another preferred embodiment, the present invention relates to theuse of (i) an enzyme capable of enzymatically converting3-methylcrotonic acid into isobutene (step I as shown in FIG. 1); and(ii) an enzyme capable of enzymatically converting 3-hydroxyisovalerate(HIV) into 3-methylcrotonic acid (step II as shown in FIG. 1); an enzymecapable of enzymatically condensing acetone and acetyl-CoA into3-hydroxyisovalerate (HIV) (step III as shown in FIG. 1), an enzymecapable of enzymatically converting acetoacetate into acetone (step IVas shown in FIG. 1), an enzyme capable of converting acetoacetyl-CoAinto acetoacetate (step Va or Vb as shown in FIG. 1) and an enzymecapable of enzymatically converting acetyl-CoA into acetoacetyl-CoAcomprising (a) (i) an enzyme capable of converting acetyl-CoA intomalonyl-CoA (step XIV as shown in FIG. 1); and (ii) an enzyme capable ofcondensing malonyl-CoA and acetyl-CoA into acetoacetyl-CoA (step XV asshown in FIG. 1); or (b) an enzyme capable of directly condensing twomolecules of acetyl-CoA into acetoacetyl-CoA (step XIII as shown inFIG. 1) for the production of isobutene.

As regards the above-mentioned enzymes as well as preferred embodimentsof said enzymes, the same applies to the use of the recombinant organismor microorganism for the production of isobutene as has been set forthabove for the methods and recombinant organisms or microorganismsaccording to the present invention.

The present invention furthermore relates to the use of (i) an enzymecapable of enzymatically converting 3-methylcrotonic acid into isobutene(step I as shown in FIG. 1); and (ii) an enzyme capable of enzymaticallyconverting 3-methylcrotonyl-CoA into 3-methylcrotonic acid (step VIa,VIb or VIc as shown in FIG. 1) for the production of isobutene.

In another preferred embodiment, the present invention relates to theuse of (i) an enzyme capable of enzymatically converting3-methylcrotonic acid into isobutene (step I as shown in FIG. 1); and(ii) an enzyme capable of enzymatically converting 3-methylcrotonyl-CoAinto 3-methylcrotonic acid (step VIa, VIb or VIc as shown in FIG. 1) andan enzyme capable of enzymatically converting 3-methylglutaconyl-CoAinto 3-methylcrotonyl-CoA (step VII as shown in FIG. 1) for theproduction of isobutene.

In another preferred embodiment, the present invention relates to theuse of (i) an enzyme capable of enzymatically converting3-methylcrotonic acid into isobutene (step I as shown in FIG. 1); and(ii) an enzyme capable of enzymatically converting 3-methylcrotonyl-CoAinto 3-methylcrotonic acid (step VIa, VIb or VIc as shown in FIG. 1); anenzyme capable of enzymatically converting 3-methylglutaconyl-CoA into3-methylcrotonyl-CoA (step VII as shown in FIG. 1) and an enzyme capableof enzymatically converting 3-hydroxy-3-methylglutaryl-CoA into3-methylglutaconyl-CoA (step VIII as shown in FIG. 1) for the productionof isobutene.

In another preferred embodiment, the present invention relates to theuse of (i) an enzyme capable of enzymatically converting3-methylcrotonic acid into isobutene (step I as shown in FIG. 1); and(ii) an enzyme capable of enzymatically converting 3-methylcrotonyl-CoAinto 3-methylcrotonic acid (step VIa, VIb or VIc as shown in FIG. 1); anenzyme capable of enzymatically converting 3-methylglutaconyl-CoA into3-methylcrotonyl-CoA (step VII as shown in FIG. 1); an enzyme capable ofenzymatically converting 3-hydroxy-3-methylglutaryl-CoA into3-methylglutaconyl-CoA (step VIII as shown in FIG. 1) and an enzymecapable of enzymatically condensing acetoacetyl-CoA and acetyl-CoA into3-hydroxy-3-methylglutaryl-CoA (step IX as shown in FIG. 1) for theproduction of isobutene.

In another preferred embodiment, the present invention relates to theuse of (i) an enzyme capable of enzymatically converting3-methylcrotonic acid into isobutene (step I as shown in FIG. 1); and(ii) an enzyme capable of enzymatically converting 3-methylcrotonyl-CoAinto 3-methylcrotonic acid (step VIa, VIb or VIc as shown in FIG. 1); anenzyme capable of enzymatically converting 3-methylglutaconyl-CoA into3-methylcrotonyl-CoA (step VII as shown in FIG. 1); an enzyme capable ofenzymatically converting 3-hydroxy-3-methylglutaryl-CoA into3-methylglutaconyl-CoA (step VIII as shown in FIG. 1); an enzyme capableof enzymatically condensing acetoacetyl-CoA and acetyl-CoA into3-hydroxy-3-methylglutaryl-CoA (step IX as shown in FIG. 1) and anenzyme capable of enzymatically converting acetyl-CoA intoacetoacetyl-CoA comprising (a) (i) an enzyme capable of convertingacetyl-CoA into malonyl-CoA (step XIV as shown in FIG. 1); and (ii) anenzyme capable of condensing malonyl-CoA and acetyl-CoA intoacetoacetyl-CoA (step XV as shown in FIG. 1); or (b) an enzyme capableof directly condensing two molecules of acetyl-CoA into acetoacetyl-CoA(step XIII as shown in FIG. 1) for the production of isobutene.

As regards the above-mentioned enzymes as well as preferred embodimentsof said enzymes, the same applies to the use of the recombinant organismor microorganism for the production of isobutene as has been set forthabove for the mehtods and recombinant organisms or microorganismsaccording to the present invention.

The present invention furthermore relates to the use of an enzymecatalyzing the enzymatic conversion of 3-methyl-3-butenoic acid intoisobutene for the production of isobutene from 3-methyl-3-butenoic acid.

The present invention furthermore relates to the use of (i) an enzymecapable of enzymatically converting 3-methyl-3-butenoic acid intoisobutene (step XVI as shown in FIG. 1) and (ii) an enzyme capable ofenzymatically converting 3-methyl-3-butenoyl-CoA into3-methyl-3-butenoic acid (step XVII as shown in FIG. 1) for theproduction of isobutene.

In another preferred embodiment, the present invention relates to theuse of (i) an enzyme capable of enzymatically converting3-methyl-3-butenoic acid into isobutene (step XVI as shown in FIG. 1)and (ii) an enzyme capable of enzymatically converting3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoic acid (step XVII asshown in FIG. 1) an enzyme capable of enzymatically converting3-methylglutaconyl-CoA into 3-methyl-3-butenoyl-CoA (step XVIII as shownin FIG. 1) for the production of isobutene.

In another preferred embodiment, the present invention relates to theuse of (i) an enzyme capable of enzymatically converting3-methyl-3-butenoic acid into isobutene (step XVI as shown in FIG. 1)and (ii) an enzyme capable of enzymatically converting3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoic acid (step XVII asshown in FIG. 1), an enzyme capable of enzymatically converting3-methylglutaconyl-CoA into 3-methyl-3-butenoyl-CoA (step XVIII as shownin FIG. 1) and an enzyme capable of enzymatically converting3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA (step VIII asshown in FIG. 1) for the production of isobutene.

In another preferred embodiment, the present invention relates to theuse of (i) an enzyme capable of enzymatically converting3-methyl-3-butenoic acid into isobutene (step XVI as shown in FIG. 1)and (ii) an enzyme capable of enzymatically converting3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoic acid (step XVII asshown in FIG. 1); an enzyme capable of enzymatically converting3-methylglutaconyl-CoA into 3-methyl-3-butenoyl-CoA (step XVIII as shownin FIG. 1); an enzyme capable of enzymatically converting3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA (step VIII asshown in FIG. 1) and an enzyme capable of enzymatically condensingacetoacetyl-CoA and acetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA (stepIX as shown in FIG. 1) for the production of isobutene.

In another preferred embodiment, the present invention relates to theuse of (i) an enzyme capable of enzymatically converting3-methyl-3-butenoic acid into isobutene (step XVI as shown in FIG. 1)and (ii) an enzyme capable of enzymatically converting3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoic acid (step XVII asshown in FIG. 1); an enzyme capable of enzymatically converting3-methylglutaconyl-CoA into 3-methyl-3-butenoyl-CoA (step XVIII as shownin FIG. 1); an enzyme capable of enzymatically converting3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA (step VIII asshown in FIG. 1); an enzyme capable of enzymatically condensingacetoacetyl-CoA and acetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA (stepIX as shown in FIG. 1) and an enzyme capable of enzymatically convertingacetyl-CoA into acetoacetyl-CoA comprising (a) (i) an enzyme capable ofconverting acetyl-CoA into malonyl-CoA (step XIV as shown in FIG. 1);and (ii) an enzyme capable of condensing malonyl-CoA and acetyl-CoA intoacetoacetyl-CoA (step XV as shown in FIG. 1); or (b) an enzyme capableof directly condensing two molecules of acetyl-CoA into acetoacetyl-CoA(step XIII as shown in FIG. 1) for the production of isobutene.

As regards the above-mentioned enzymes as well as preferred embodimentsof said enzymes, the same applies to the use of the recombinant organismor microorganism for the production of isobutene as has been set forthabove for the mehtods and recombinant organisms or microorganismsaccording to the present invention.

In a further aspect, the present invention relates to any of the aboveuses of enzymes for the production of isobutene, wherein additionally

-   -   a) an enzyme capable of enzymatically converting        3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid with a        concomitant transfer of CoA from 3-methylcrotonyl-CoA on        3-hydroxyisovalerate (HIV) to result in 3-hydroxyisovaleryl-CoA        (step Xa as schematically shown in FIG. 19); and/or    -   b) an enzyme capable of enzymatically converting        3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA (step Xb        as schematically shown in FIG. 20); and/or    -   c) an enzyme capable of enzymatically converting        3-hydroxyisovaleryl-CoA into 3-methylcrotonyl-CoA (step XI as        schematically shown in FIG. 21); and/or    -   d) an enzyme capable of enzymatically converting        3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA (step        XII as schematically shown in FIG. 22)

as described herein above is used.

As regards the above-mentioned enzymes as well as preferred embodimentsof said enzymes, the same applies to the recombinant organism ormicroorganism as has been set forth above for the methods according tothe present invention.

Furthermore, the present invention relates to a composition comprising3-methylcrotonic acid and a recombinant organism or microorganism,wherein said recombinant organism or microorganism expresses (i) anenzyme capable of enzymatically converting 3-methylcrotonic acid intoisobutene (step I as shown in FIG. 1); and/or (ii) an enzyme capable ofenzymatically converting 3-hydroxyisovalerate (HIV) into3-methylcrotonic acid (step II as shown in FIG. 1), and/or which furtherexpresses an enzyme capable of enzymatically condensing acetone andacetyl-CoA into 3-hydroxyisovalerate (HIV) (step III as shown in FIG.1), and/or which further expresses an enzyme capable of enzymaticallyconverting acetoacetate into acetone (step IV as shown in FIG. 1),and/or which further expresses an enzyme capable of convertingacetoacetyl-CoA into acetoacetate (step Va or Vb as shown in FIG. 1)and/or which further expresses an enzyme capable of enzymaticallyconverting acetyl-CoA into acetoacetyl-CoA comprising (a) (i) an enzymecapable of converting acetyl-CoA into malonyl-CoA (step XIV as shown inFIG. 1); and (ii) an enzyme capable of condensing malonyl-CoA andacetyl-CoA into acetoacetyl-CoA (step XV as shown in FIG. 1); or (b) anenzyme capable of directly condensing two molecules of acetyl-CoA intoacetoacetyl-CoA (step XIII as shown in FIG. 1).

Furthermore, the present invention relates to a composition comprising3-methylcrotonic acid (i) an enzyme capable of enzymatically converting3-methylcrotonic acid into isobutene (step I as shown in FIG. 1); and/or(ii) an enzyme capable of enzymatically converting 3-hydroxyisovalerate(HIV) into 3-methylcrotonic acid (step II as shown in FIG. 1); and/or anenzyme capable of enzymatically condensing acetone and acetyl-CoA into3-hydroxyisovalerate (HIV) (step III as shown in FIG. 1), and/or anenzyme capable of enzymatically converting acetoacetate into acetone(step IV as shown in FIG. 1), and/or an enzyme capable of convertingacetoacetyl-CoA into acetoacetate (step Va or Vb as shown in FIG. 1)and/or an enzyme capable of enzymatically converting acetyl-CoA intoacetoacetyl-CoA comprising (a) (i) an enzyme capable of convertingacetyl-CoA into malonyl-CoA (step XIV as shown in FIG. 1); and (ii) anenzyme capable of condensing malonyl-CoA and acetyl-CoA intoacetoacetyl-CoA (step XV as shown in FIG. 1); or (b) an enzyme capableof directly condensing two molecules of acetyl-CoA into acetoacetyl-CoA(step XIII as shown in FIG. 1).

As regards the above-mentioned enzymes as well as preferred embodimentsof said enzymes, the same applies to the use of the recombinant organismor microorganism for the production of isobutene as has been set forthabove for the methods and recombinant organisms or microorganismsaccording to the present invention.

Furthermore, the present invention relates to a composition comprising3-methylcrotonic acid and a recombinant organism or microorganism,wherein said recombinant organism or microorganism expresses (i) anenzyme capable of enzymatically converting 3-methylcrotonic acid intoisobutene (step I as shown in FIG. 1); and/or (ii) an enzyme capable ofenzymatically converting 3-methylcrotonyl-CoA into 3-methylcrotonic acid(step VIa, VIb or VIc as shown in FIG. 1), and/or which furtherexpresses an enzyme capable of enzymatically converting3-methylglutaconyl-CoA into 3-methylcrotonyl-CoA (step VII as shown inFIG. 1), and/or which further expresses an enzyme capable ofenzymatically converting 3-hydroxy-3-methylglutaryl-CoA into3-methylglutaconyl-CoA (step VIII as shown in FIG. 1), and/or whichfurther expresses an enzyme capable of enzymatically condensingacetoacetyl-CoA and acetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA (stepIX as shown in FIG. 1) and/or which further expresses an enzyme capableof enzymatically converting acetyl-CoA into acetoacetyl-CoA comprising(a) (i) an enzyme capable of converting acetyl-CoA into malonyl-CoA(step XIV as shown in FIG. 1); and (ii) an enzyme capable of condensingmalonyl-CoA and acetyl-CoA into acetoacetyl-CoA (step XV as shown inFIG. 1); or (b) an enzyme capable of directly condensing two moleculesof acetyl-CoA into acetoacetyl-CoA (step XIII as shown in FIG. 1).

Furthermore, the present invention relates to a composition comprising3-methylcrotonic acid and (i) an enzyme capable of enzymaticallyconverting 3-methylcrotonic acid into isobutene (step I as shown in FIG.1); and/or (ii) an enzyme capable of enzymatically converting3-methylcrotonyl-CoA into 3-methylcrotonic acid (step VIa, VIb or VIc asshown in FIG. 1); and/or an enzyme capable of enzymatically converting3-methylglutaconyl-CoA into 3-methylcrotonyl-CoA (step VII as shown inFIG. 1); and/or an enzyme capable of enzymatically converting3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA (step VIII asshown in FIG. 1); and/or an enzyme capable of enzymatically condensingacetoacetyl-CoA and acetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA (stepIX as shown in FIG. 1) and/or an enzyme capable of enzymaticallyconverting acetyl-CoA into acetoacetyl-CoA comprising (a) (i) an enzymecapable of converting acetyl-CoA into malonyl-CoA (step XIV as shown inFIG. 1); and (ii) an enzyme capable of condensing malonyl-CoA andacetyl-CoA into acetoacetyl-CoA (step XV as shown in FIG. 1); or (b) anenzyme capable of directly condensing two molecules of acetyl-CoA intoacetoacetyl-CoA (step XIII as shown in FIG. 1).

As regards the above-mentioned enzymes as well as preferred embodimentsof said enzymes, the same applies to the use of the recombinant organismor microorganism for the production of isobutene as has been set forthabove for the mehtods and recombinant organisms or microorganismsaccording to the present invention.

Furthermore, the present invention relates to a composition comprising3-methyl-3-butenoic acid and a recombinant organism or microorganism,wherein said recombinant organism or microorganism expresses (i) anenzyme capable of enzymatically converting 3-methyl-3-butenoic acid intoisobutene (step XVI as shown in FIG. 1) and/or (ii) an enzyme capable ofenzymatically converting 3-methyl-3-butenoyl-CoA into3-methyl-3-butenoic acid (step XVII as shown in FIG. 1), and/or whichfurther expresses an enzyme capable of enzymatically converting3-methylglutaconyl-CoA into 3-methyl-3-butenoyl-CoA (step XVIII as shownin FIG. 1), and/or which further expresses an enzyme capable ofenzymatically converting 3-hydroxy-3-methylglutaryl-CoA into3-methylglutaconyl-CoA (step VIII as shown in FIG. 1), and/or whichfurther expresses an enzyme capable of enzymatically condensingacetoacetyl-CoA and acetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA (stepIX as shown in FIG. 1) and/or which further expresses an enzyme capableof enzymatically converting acetyl-CoA into acetoacetyl-CoA comprising(a) (i) an enzyme capable of converting acetyl-CoA into malonyl-CoA(step XIV as shown in FIG. 1); and (ii) an enzyme capable of condensingmalonyl-CoA and acetyl-CoA into acetoacetyl-CoA (step XV as shown inFIG. 1); or (b) an enzyme capable of directly condensing two moleculesof acetyl-CoA into acetoacetyl-CoA (step XIII as shown in FIG. 1).

Furthermore, the present invention relates to a composition comprising3-methyl-3-butenoic acid and (i) an enzyme capable of enzymaticallyconverting 3-methyl-3-butenoic acid into isobutene (step XVI as shown inFIG. 1) and/or (ii) an enzyme capable of enzymatically converting3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoic acid (step XVII asshown in FIG. 1); and/or an enzyme capable of enzymatically converting3-methylglutaconyl-CoA into 3-methyl-3-butenoyl-CoA (step XVIII as shownin FIG. 1); and/or an enzyme capable of enzymatically converting3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA (step VIII asshown in FIG. 1); and/or an enzyme capable of enzymatically condensingacetoacetyl-CoA and acetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA (stepIX as shown in FIG. 1) and/or an enzyme capable of enzymaticallyconverting acetyl-CoA into acetoacetyl-CoA comprising (a) (i) an enzymecapable of converting acetyl-CoA into malonyl-CoA (step XIV as shown inFIG. 1); and (ii) an enzyme capable of condensing malonyl-CoA andacetyl-CoA into acetoacetyl-CoA (step XV as shown in FIG. 1); or (b) anenzyme capable of directly condensing two molecules of acetyl-CoA intoacetoacetyl-CoA (step XIII as shown in FIG. 1) for the production ofisobutene.

As regards the above-mentioned enzymes as well as preferred embodimentsof said enzymes, the same applies to the use of the recombinant organismor microorganism for the production of isobutene as has been set forthabove for the mehtods and recombinant organisms or microorganismsaccording to the present invention.

In a further aspect, the present invention relates to any of theabove-described compositions, wherein the organism or microorganism isan organism or microorganism which additionally further expresses

-   -   a) an enzyme capable of enzymatically converting        3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid with a        concomitant transfer of CoA from 3-methylcrotonyl-CoA on        3-hydroxyisovalerate (HIV) to result in 3-hydroxyisovaleryl-CoA        (step Xa as schematically shown in FIG. 19); and/or    -   b) an enzyme capable of enzymatically converting        3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA (step Xb        as schematically shown in FIG. 20); and/or    -   c) an enzyme capable of enzymatically converting        3-hydroxyisovaleryl-CoA into 3-methylcrotonyl-CoA (step XI as        schematically shown in FIG. 21); and/or    -   d) an enzyme capable of enzymatically converting        3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA (step        XII as schematically shown in FIG. 22)

as described herein above.

In a further aspect, the present invention relates to any of theabove-described compositions which further additionally comprises

-   -   a) an enzyme capable of enzymatically converting        3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid with a        concomitant transfer of CoA from 3-methylcrotonyl-CoA on        3-hydroxyisovalerate (HIV) to result in 3-hydroxyisovaleryl-CoA        (step Xa as schematically shown in FIG. 19); and/or    -   b) and enzyme capable of enzymatically converting        3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA (step Xb        as schematically shown in FIG. 20); and/or    -   c) an enzyme capable of enzymatically converting        3-hydroxyisovaleryl-CoA into 3-methylcrotonyl-CoA (step XI as        schematically shown in FIG. 21); and/or    -   d) an enzyme capable of enzymatically converting        3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA (step        XII as schematically shown in FIG. 22) as described herein        above.

As regards the above-mentioned enzymes as well as preferred embodimentsof said enzymes, the same applies to the recombinant organism ormicroorganism as has been set forth above for the methods according tothe present invention.

FIG. 1: shows an artificial pathway for isobutene production fromacetyl-CoA via 3-methylcrotonic acid. Moreover, enzymatic recycling ofmetabolites which may occur during the pathway are shown in steps Xa,Xb, XI and XII.

FIG. 2A: Schematic reaction of the enzymatic prenylation of a flavinmononucleotide (FMN) into the corresponding modified (prenylated) flavincofactor.

FIG. 2B: Schematic reaction of the enzymatic conversion of3-methylcrotonic acid into isobutene.

FIG. 3: Schematic reaction of the enzymatic conversion of3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid.

FIG. 4: Schematic reaction of the enzymatic condensation of acetyl-CoAand acetone into 3-hydroxyisovalerate.

FIG. 5: Schematic reaction of the enzymatic conversion of acetoacetateinto acetone.

FIG. 6: Schematic reaction of the enzymatic conversion ofacetoacetyl-CoA into acetoacetate by hydrolysing the CoA thioester ofacetoacetyl-CoA resulting in acetoacetate.

FIG. 7: Schematic reaction of the enzymatic conversion ofacetoacetyl-CoA into acetoacetate by transferring the CoA group ofacetoacetyl-CoA on acetate, resulting in the formation of acetoacetateand acetyl-CoA.

FIG. 8: Schematic reaction of the enzymatic conversion of3-methylcrotonyl-CoA into 3-methylcrotonic acid.

FIG. 9: Schematic reaction of the enzymatic conversion of3-methylcrotonyl-CoA into 3-methylcrotonic acid via step VIa as shown inFIG. 1.

FIG. 10: Schematic reaction of the enzymatic conversion of3-methylcrotonyl-CoA into 3-methylcrotonic acid via step VIb as shown inFIG. 1.

FIG. 11: Schematic reaction of the enzymatic conversion of3-methylcrotonyl-CoA into 3-methylcrotonic acid via step VIc as shown inFIG. 1.

FIG. 12: Schematic reaction of the enzymatic conversion of3-methylglutaconyl-CoA into 3-methylcrotonyl-CoA.

FIG. 13: Schematic reaction of the enzymatic conversion of3-hydroxy-3-methylglutaryl-CoA into 3-methylglutaconyl-CoA.

FIG. 14: Schematic reaction of the enzymatic condensation of acetylCoAand acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA.

FIG. 15: Schematic reaction of the enzymatic condensation of twomolecules of acetyl-CoA into acetoacetyl-CoA.

FIG. 16: Schematic reaction of the enzymatic conversion of acetyl-CoAinto malonyl-CoA.

FIG. 17: Schematic reaction of the enzymatic condensation of malonyl-CoAand acetyl-CoA into acetoacetyl-CoA.

FIG. 18: shows enzymatic recycling steps of metabolites (steps Xa, Xb,XI and XII as also shown in FIG. 1) which may occur during the pathwayof isobutene production from acetyl-CoA via 3-methylcrotonic acid.

FIG. 19: Schematic reaction of the enzymatic conversion of3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid with a concomitanttransfer of CoA from 3-methylcrotonyl-CoA on 3-hydroxyisovalerate (HIV)to result in 3-hydroxyisovaleryl-CoA.

FIG. 20: Schematic reaction of the enzymatic conversion of3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA.

FIG. 21: Schematic reaction of the enzymatic conversion of3-hydroxyisovaleryl-CoA into 3-methylcrotonyl-CoA.

FIG. 22: Schematic reaction of the general enzymatic conversion of3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA.

FIG. 23: Schematic reaction of the enzymatic conversion of3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA via3-hydroxyisovaleryl-adenosine monophosphate.

FIG. 24: Schematic reaction of the enzymatic conversion of3-hydroxyisovalerate (HIV) into 3-hydroxyisovaleryl-CoA via3-hydroxyisovaleryl phosphate.

FIG. 25: Schematic reaction of the enzymatic conversion of3-methyl-3-butenoic acid into isobutene.

FIG. 26: Schematic reaction of the enzymatic conversion of3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoic acid.

FIG. 27: Schematic reaction of the enzymatic conversion of3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoic acid by making use of aCoA-transferase.

FIG. 28: Schematic reaction of the enzymatic conversion of3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoic acid by making use of athioester hydrolase.

FIG. 29: Schematic reaction of the enzymatic conversion of3-methyl-3-butenoyl-CoA into 3-methyl-3-butenoic acid in a two-stepreaction via 3-methyl-3-butenoyl phosphate.

FIG. 30: Schematic reaction of the enzymatic conversion of3-methylglutaconyl-CoA into 3-methyl-3-butenoyl-CoA.

FIG. 31: Structure of a phosphopantetheine moiety.

FIG. 32: Schematic illustration for the conversion of3-methylcrotonyl-CoA into 3-methylcrotonic acid via 3-methylbutyryl-CoAand 3-methylbutyric acid.

FIG. 33: shows an overlay of typical GC-chromatograms obtained for thecatalytic assay of UbiD protein from Saccharomyces cerevisiae with thecorresponding controls as outlined in Example 2.

FIG. 34A: shows an overlay of typical HPLC-chromatograms (analysis of3-methylcrotonyl-CoA, 3-methylcrotonic acid and CoA-SH) obtained for the“Enzymatic assay” (assay A, Example 3) and the “Enzyme-free assay”(assay H, Example 3). The consumption of 3-methylcrotonyl-CoA withsimultaneous production of CoA-SH and 3-methylcrotonic acid was observedin the enzymatic assay combining phosphate butyryltransferase withbutyrate kinase.

FIG. 34B: shows an overlay of typical HPLC-chromatograms (analysis ofADP and ATP) obtained for the “Enzymatic assay” (assay A, Example 3) andthe “Enzyme-free assay” (assay H, Example 3). The consumption of ADPwith simultaneous production of ATP was observed in the enzymatic assaycombining phosphate butyryltransferase with butyrate kinase.

FIG. 35: shows the results of the production of 3-methylcrotonic acidand ATP in the enzymatic assays, comprising phosphate butyryltransferasefrom Bacillus subtilis combined with different butyrate kinases.Moreover, the production of 3-methylcrotonic acid and ATP in controlassays is shown.

FIG. 36: shows the results of the production of 3-methylcrotonic acidand ATP in the enzymatic assays, comprising phosphate butyryltransferasefrom from Enterococcus faecalis combined with different butyratekinases. Moreover, the production of 3-methylcrotonic acid and ATP indifferent control assays is shown.

FIG. 37: shows an example of typical HPLC-chromatogram obtained for theenzymatic assay with acyl-CoA thioesterase II from Pseudomonas putida asoutlined in Example 5.

FIG. 38: shows an overlay of typical chromatograms obtained for theproduction of isobutene from 3-methylcrotonic in a recombinant E. colistrain overexpressing UbiD protein from Saccharomyces cerevisiae andUbiX protein from Escherichia coli (strain A) or overexpressing UbiDprotein from Saccharomyces cerevisiae alone (strain B) or carrying anempty vector (negative control, strain C).

FIG. 39: shows an overlay of typical chromatograms obtained for theproduction of isobutene from 3-methylcrotonyl-CoA in the one potenzymatic assay as outlined in Example 7, and the correspondingcontrols.

FIGS. 40A and 40B: shows chromatograms for enzymatic assays (FIG. 40A)and control assays (FIG. 40B). A significant quantity of acetyl-CoA and3-methylcrotonic acid was produced from acetate and 3-methylcrotonyl-CoAin the presence of Co-A transferase (FIG. 40A) while no product wasobserved in the control assay without enzyme (FIG. 40B).

FIG. 41: shows 3-methylglutaconyl-CoA (MG-CoA) peak areas obtained fromHPLC-based analysis.

FIG. 42: Metabolic pathway for the biosynthesis of isobutene fromacetyl-CoA via 3-methycrotonic acid, implemented in Escherichia coli.

In this specification, a number of documents including patentapplications are cited. The disclosure of these documents, while notconsidered relevant for the patentability of this invention, is herewithincorporated by reference in its entirety. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

The invention will now be described by reference to the followingexamples which are merely illustrative and are not to be construed as alimitation of the scope of the present invention.

EXAMPLES General Methods and Materials

All reagents and materials used in the experiences were obtained fromSigma-Aldrich Company (St. Louis, Mo.) unless otherwise specified.Materials and methods suitable for growth of bacterial cultures andprotein expression are well known in the art.

Example 1 Gene Synthesis, Cloning and Expression of Recombinant Proteins

The sequences of the studied enzymes were generated by oligonucleotideconcatenation to fit the codon usage of E. coli (genes were commerciallysynthesized by GeneArt®). A stretch of 6 histidine codons was insertedafter the methionine initiation codon to provide an affinity tag forpurification. The gene thus synthesized was cloned in a pET-25b (+)expression vector (vectors were constructed by GeneArt®). Vector pCANcontained gene coding for UbiX protein (3-octaprenyl-4-hydroxybenzoatecarboxy-lyase partner protein) from Escherichia coli (Uniprot AccessionNumber: P0AG03) was purchased from NAIST (Nara Institute of Science andTechnology, Japan, ASKA collection). Provided vector contained a stretchof 6 histidine codons after the methionine initiation codon.

Competent E. coli BL21 (DE3) cells (Novagen) were transformed with thesevectors according to standard heat shock procedure. The transformedcells were grown with shaking (160 rpm) using ZYM-5052 auto-inductionmedium (Studier F W, Prot. Exp. Pur. 41, (2005), 207-234) for 6 h at 30°C. and protein expression was continued at 18° C. overnight(approximately 16 h). For the recombinant strain over-expressing UbiXfrom E. coli, 500 μM of Flavin Mononucleotide (FMN) were added to thegrowth medium. The cells were collected by centrifugation at 4° C.,10,000 rpm for 20 min and the pellets were stored at −80° C.

Protein Purification and Concentration

The pellets from 200 ml of cultured cells were thawed on ice andresuspended in 6 ml of 50 mM Tris-HCl buffer pH 7.5 containing 100 mMNaCl in the case of the recombinant strain overexpressing UbiX proteinand in 6 ml of 50 mM Tris-HCl buffer pH 7.5, 10 mM MgCl₂, 10 mMimidazole and 5 mM DTT in the case of the recombinant strainoverexpressing UbiD protein. Twenty microliters of lysonase (Novagen)were added. Cells were then incubated 10 min at room temperature,returned to ice for 20 min and the lysis was completed by sonication3×15 seconds. The cellular lysate contained UbiX protein was reserved onice. The bacterial extracts contained UbiD proteins were then clarifiedby centrifugation at 4° C., 4000 rpm for 40 min. The clarified bacteriallysates were loaded onto a PROTINO-2000 Ni-TED column (Macherey-Nagel)allowing adsorption of 6-His tagged proteins. Columns were washed andthe enzymes of interest were eluted with 6 ml of 100 mM Tris-HCl bufferpH 7.5 containing 100 mM NaCl and 250 mM imidazole. Eluates were thenconcentrated, desalted on Amicon Ultra-4 10 kDa filter unit (Millipore)and enzymes were resuspended in 50 mM Tris-HCl buffer pH 7.5, containing50 mM NaCl and 5 mM DTT.

The purity of proteins thus purified varied from 80% to 90% as estimatedby SDS-PAGE analysis. Protein concentration was determined by direct UV280 nm measurement on the NanoDrop 1000 spectrophotometer (ThermoScientific) and by Bradford assay (BioRad).

Example 2 In Vitro Decarboxylation of 3-Methylcrotonic Acid intoIsobutene Catalyzed by an Association of Lysate, Containing UbiXProtein, with Purified UbiD Protein

0.5 M stock solution of 3-methylcrotonic acid was prepared in water andadjusted to pH 7.0 with 10 M solution of NaOH.

Two UbiD proteins (Table C) were purified according to the proceduredescribed in Example 1.

Enzymatic assays were carried out in 2 ml glass vials (Interchim) underthe following conditions:

50 mM Tris-HCl buffer pH 7.5

20 mM NaCl

10 mM MgCl₂

5 mM DTT

50 mM 3-methylcrotonic acid

1 mg/ml purified UbiD protein

50 μl lysate contained UbiX protein

Total volume of the assays were 300 μl.

A series of control assays were performed in parallel (Table C).

The vials were sealed and incubated for 120 min at 30° C. The assayswere stopped by incubating for 2 min at 80° C. and the isobutene formedin the reaction headspace was analysed by Gas Chromatography (GC)equipped with Flame Ionization Detector (FID).

For the GC analysis, one ml of the headspace gas was separated in aBruker GC-450 system equipped with a GS-alumina column (30 m×0.53 mm)(Agilent) using isothermal mode at 130° C. Nitrogen was used as carriergas with a flow rate of 6 ml/min.

The enzymatic reaction product was identified by comparison with anisobutene standard. Under these GC conditions, the retention time ofisobutene was 2.42 min.

A significant production of isobutene from 3-methylcrotonic acid wasobserved in the combined assays (UbiD protein+UbiX protein). Incubationof lysate containing UbIX protein alone did not result in isobuteneproduction. These data indicate that the two enzymes present in theassays cooperated to perform the decarboxylation of 3-methylcrotonicacid into isobutene. A typical chromatogram obtained in the assay withUbiD protein from Saccharomyces cerevisiae is shown on FIG. 33.

TABLE C Isobutene production, arbitrary Assay composition units UbiDprotein from C. dubliniensis 470 (Uniprot Acession Number: B9WJ66) +lysate contained UbiX protein from E. coli + substrate UbiD protein fromC. dubliniensis (Uniprot 9.2 Acession Number: B9WJ66) + substrate UbiDprotein from S. cervisiae (Uniprot 1923 Acession Number : Q03034) +lysate contained UbiX protein from E. coli + substrate UbiD protein fromS. cerivisae (Uniprot 31 Acession Number: Q03034) + substrate Lysatecontained UbiX protein 0 from E. coli + substrate “No substratecontrol”: UbiD protein from 0 C. dubliniensis (Uniprot Acession Number:B9WJ66) + lysate contained UbiX protein from E. coli, without substrate“No substrate control”: UbiD protein 0 from S. cervisiae (UniprotAcession Number : Q03034) + lysate contained UbiX protein from E. coli,without substrate

Example 3 Conversion of 3-Methylcrotonyl-CoA and ADP into3-Methylcrotonic Acid and ATP Catalysed by the Combined Action ofPhosphate Butyryltransferase from Bacillus subtilis and Butyrate Kinasefrom Lactobacillus casei or Geobacillus sp

The corresponding enzymes were obtained and purified according to theprocedure described in Example 1.

The enzymatic assays were conducted in a total reaction volume of 0.2 mlThe standard reaction mixture contained:

50 mM potassium phosphate buffer pH 7.5

4 mM 3-methylcrotonyl-CoA

4 mM ADP

10 mM MgCl₂

10 mM NaCl

0.2 mg/ml purified phosphate butyryltransferase from Bacillus subtilis(Uniprot Accession Number: P54530)

0.2 mg/ml purified butyrate kinase from Lactobacillus casei (UniprotAccession Number: K0N529) or Geobacillus sp. (Uniprot accession number:L8A0E1).

A series of controls were performed in parallel (Assays C-H Table D).

TABLE D Assay composition A B C D E F G H3-methylcrotonyl-CoA + + + + + + + + ADP + + + + + + phosphatebutyryltransferase + + + + + from Bacillus subtilis butyrate kinasefrom + + + Lactobacillus casei butyrate kinase from + + + Geobacillus sp

Assays were incubated for 20 min with shaking at 30° C.

After an incubation period, the reactions were stopped by heating thereaction medium 4 min at 90° C. The samples were centrifuged, filteredthrough a 0.22 μm filter and the clarified supernatants were transferredinto a clean vial for the further analysis. The consumption of ADP and3-methylcrotonyl-CoA, and the formation of ATP, 3-methylcrotonic acidand free coenzyme A (CoA-SH) were followed by using HPLC-based methods.

HPLC-Based Analysis of ADP and ATP

HPLC analysis was performed using 1260 Inifinity LC System (Agilent),equipped with column heating module and RI detector. 2 μl of sampleswere separated on Polaris C18-A column (150×4.6 mm, 5 μm particle size,column temp. 30° C.) with a mobile phase flow rate of 1.5 ml/min. Theseparation was performed using 8.4 mM sulfuric acid in H₂O/MeOH mixedsolution (99/1) (V/V). In these conditions, the retention time of ADPand ATP were 2.13 min and 2.33 min, respectively.

HPLC Based Analysis of 3-Methylcrotonyl-CoA, 3-Methylcrotonic Acid andFree Coenzyme A (CoA-SH)

HPLC analysis was performed using 1260 Inifinity LC System (Agilent),equipped with column heating module and UV detector (260 nm). 1 μl ofsamples were separated on Zorbax SB-Aq column (250×4.6 mm, 5 μm particlesize, column temp. 30° C.), with a mobile phase flow rate of 1.5 ml/min.The separation was performed using mixed A (H₂O containing 8.4 mMsulfuric acid) and B (acetonitrile) solutions in a linear gradient (0% Bat initial time 0 min→70% B at 8 min). In these conditions, theretention time of 3-methylcrotonyl-CoA, 3-methylcrotonic acid and freecoenzyme A (CoA-SH) were 5.38 min, 5.73 min and 4.07 min, respectively.

Typical chromatograms obtained for the enzymatic assay A and enzyme-freeassay H are shown on FIGS. 34A and 34B.

The results of HPLC analysis are summarized in FIG. 35.

The obtained data indicate that 3-methylcrotonyl-CoA was converted into3-methylcrotonic acid with the concomitant generation of ATP from ADP ina two-step reaction, catalyzed respectively by two enzymes (assays A andB). Thus, the conversion occurred through the formation of theintermediate 3-methylcrotonyl phosphate followed by the transfer ofphosphate group from this intermediate on ADP thereby releasing ATP.

A certain quantity of 3-methylcrotonic acid was produced withoutsimultaneous generation of ATP, when phosphate butyryltransferase wasused alone (assay E). This production is due to a spontaneous hydrolysisof 3-methylcrotonyl phosphate generated by the action of phosphatebutyryltransferase.

The production of 3-methylcrotonic acid was observed in the same mannerfor the control assays without ADP (assays C and D). This production wasalso due to a hydrolysis of the 3-methylcrotonyl phosphate generated bythe action of phosphate butyryltransferase.

Example 4 Conversion of 3-Methylcrotonyl-CoA and ADP into3-Methylcrotonic Acid and ATP Catalysed by the Combined Action of thePhosphate Butyryltransferase from Enterococcus faecalis and ButyrateKinase from Lactobacillus casei or Geobacillus sp

The corresponding enzymes were obtained and purified according to theprocedure described in Example 1.

The enzymatic assays were conducted in a total reaction volume of 0.2 ml

The standard reaction mixture contained:

50 mM potassium phosphate buffer pH 7.5

4 mM 3-methylcrotonyl-CoA

4 mM ADP

10 mM MgCl₂

10 mM NaCl

0.2 mg/ml purified phosphate butyryltransferase from Enterococcusfaecalis (Uniprot Accession Number: S4BZL5)

0.2 mg/ml purified butyrate kinase from Lactobacillus casei (UniprotAccession Number: K0N529) or Geobacillus sp. (Uniprot Accession Number:L8A0E1)

A series of controls were performed in parallel (Assays C-H Table E).

TABLE E Assay composition A B C D E F G H3-methylcrotonyl-CoA + + + + + + + + ADP + + + + + + phosphate + + + + +butyryltransferase from Enterococcus faecalis butyrate kinase from + + +Lactobacillus casei butyrate kinase from + + + Geobacillus sp

Assays were incubated for 20 min with shaking at 30° C.

After an incubation period, the reactions were stopped by heating thereaction medium 4 min at 90° C. The samples were centrifuged, filteredthrough a 0.22 μm filter and the clarified supernatants were transferredinto a clean vial for further analysis. The consumption of ADP and3-methylcrotonyl-CoA, and the formation of ATP and 3-methylcrotonic acidand free coenzyme A (CoA-SH) were followed by HPLC analysis according tothe methods described in Example 3.

The results of HPLC analysis are summarized in FIG. 36.

The obtained data indicate that 3-metylcrotonyl-CoA was converted into3-methylcrotonic acid with the concomitant generation of ATP from ADP ina two-step reaction, catalyzed respectively by two enzymes (assays A andB). Thus, the conversion occurred through the formation of theintermediate 3-methylcrotonyl phosphate followed by transfer ofphosphate group from this intermediate on ADP thereby releasing ATP.

A significant production of 3-methylcrotonic acid, without simultaneousgeneration of ATP, was observed when phosphate butyryltransferase wasused alone (assay E). This production was due to a hydrolysis of3-methylcrotonyl phosphate generated by the action of phosphatebutyryltransferase.

The production of 3-methylcrotonic acid was observed in the same mannerfor the control assays without ADP (assays C and D). This production wasalso due to a hydrolysis of the 3-methylcrotonyl phosphate generated bythe action of phosphate butyryltransferase.

Example 5 Enzyme-Catalyzed Hydrolysis of 3-Methylcrotonyl-CoA into3-Methylcrotonic Acid and Free Coenzyme A

The gene coding for acyl-CoA thioesterase II from Pseudomonas putida wassynthesized according to the procedure described in Example 1.

Vector pCAN contained gene encoding acyl-CoA thioesterase 2 (TesB) fromEscherichia coli were purchased from NAIST (Nara Institute of Scienceand Technology, Japan, ASKA collection). Provided vector contained astretch of 6 histidine codons after the methionine initiation codon. Thecorresponding enzymes were produced according to the procedure describedin Example 1.

The enzymatic assays were conducted in a total reaction volume of 0.2ml.

The standard reaction mixture contained:

50 mM HEPES pH 7.0

10 mM 3-methylcrotonyl-CoA

20 mM MgCl2

20 mM NaCl

1 mg/ml purified recombinant thioesterase.

Control assays were performed in which either no enzyme was added, or nosubstrate was added.

The assays were incubated for 30 min with shaking at 30° C., thereactions were stopped by the addition of 0.1 ml acetonitrile and thesamples were then analysed by HPLC-based procedure.

HPLC based analysis of the consumption of 3-methylcrotonyl-CoA and theformation of 3-methylcrotonic acid and free coenzyme A (CoA-SH)

HPLC analysis was performed using 1260 Inifinity LC System (Agilent),equipped with column heating module and UV detector (210 nm). 5 μl ofsamples were separated on Zorbax SB-Aq column (250×4.6 mm, 5 μm particlesize, column temp. 30° C.), with a mobile phase flow rate of 1.5 ml/min.The separation was performed using mixed A (H₂O containing 8.4 mMsulfuric acid) and B (acetonitrile) solutions in a linear gradient (0% Bat initial time 0 min→70% B at 8 min). Commercial 3-methylcrotonyl-CoA,3-methylcrotonic acid (Sigma-Aldrich) and CoA-SH (Sigma-Aldrich) wereused as references. In these conditions, the retention time of freecoenzyme A (CoA-SH), 3-methylcrotonyl-CoA and 3-methylcrotonic acid were4.05, 5.38 and 5.83 min, respectively.

No 3-methylcrotonic acid signal was observed in control assays.

The both studied thioesterases catalyzed the hydrolysis of3-methylcrotonyl-CoA with the formation of 3-methylcrotonic acid. Anexample of chromatogram obtained with acyl-CoA thioesterase II fromPseudomonas putida is shown on FIG. 37.

The production of 3-methylcrotonic acid observed in the enzymatic assaysare shown in Table F.

TABLE F Uniprot Accession 3-methylcrotonic Gene name Organism Numberacid produced, mM tesB Escherichia coli POAGG2 0.6 tesB Pseudomonasputida Q88DR1 3.1

Example 6 In Vivo Decarboxylation of 3-Methylcrotonic Acid IntoIsobutene Catalyzed by an Association of UbiX Protein from Escherichiacoli and UbiD Protein from Saccharomyces cerevisiae

The gene coding for UbiD protein from S. cerevisiae (Uniprot AccessionNumber: Q03034) was codon optimized for expression in E. coli andsynthesized by GeneArt® (Life Technologies). This studied gene was thenPCR amplified from the pMK-RQ vector (master plasmid provided byGeneArt) using forward primer with Ncol restriction site and a reverseprimer, containing BamHl restriction site. The gene coding for UbiXprotein from E. coli (Uniprot Accession Number: P0AG03) was amplified byPCR with a forward primer, containing Ndel restriction site and areverse primer containing Kpnl restriction site. The previouslydescribed pCAN vector (Example 1) served as template for this PCR step.These two obtained PCR products (UbiD protein and UbiX protein) werecloned into pETDuet™-1 co-expression vector (Novagen). The constructedrecombinant plasmid was verified by sequencing. Competent E. coliBL21(DE3) cells (Novagen) were transformed with this vector according tostandard heat shock procedure and plated out onto LB agar platessupplemented with ampicillin (0.1 mg/ml) (termed “strain A”).

BL21(DE3) strain transformed with pET-25b(+) vector, carrying only thegene of UbiD protein from S. cerevisae was also used in this study(termed “strain B”). BL21(DE3) strain transformed with an emptypET-25b(+) vector was used as a negative control in the subsequentassays (termed “strain C”).

Single transformants were used to inoculate LB medium, supplemented withampicillin, followed by incubation at 30° C. overnight. 1 ml of thisovernight culture was used to inoculate 300 ml of ZYM-5052 auto-inducingmedia (Studier FW (2005), local citation). The cultures were grown for20 hours at 30° C. and 160 rpm shaking.

A volume of cultures corresponding to OD600 of 30 was removed andcentrifuged. The pellet was resuspended in 30 ml of MS medium (RichaudC., Mengin-Leucreulx D., Pochet S., Johnson E J., Cohen G N. and MarHereP, The Journal of Biological Chemistry, 268, (1993), 26827-26835),containing glucose (45 g/L) and MgSO4 (1 mM) and supplemented with 10 mM3-methylcrotonic acid. These cultures were then incubated in 160 mlbottles, sealed with a screw cap, at 30° C. with shaking for 22 h. ThepH value of the cultures was adjusted to 8.5 after 8 hours of incubationby using 30% NH₄OH.

After an incubation period, the isobutene produced in the headspace wasanalysed by Gas Chromatography (GC) equipped Flame Ionization Detector(FID). One ml of the headspace gas phase was separated and analysedaccording to the method described in Example 2.

No isobutene was formed with the control strain C carrying an emptyvector. The highest production of isobutene was observed for the strainA over-expressing the both genes, UbiD protein from S. cerevisiae andUbiX protein from E. coli. A significant production of isobutene wasobserved for the strain B over-expressing UbiD protein alone. Thus,endogenous UbiX of E. coli can probably contribute to activate UbiDprotein from S. cerivisae (FIG. 38).

Example 7 One Pot Enzymatic Synthesis of Isobutene from3-Methylcrotonyl-CoA Catalyzed by an Association ofPhosphotransbutyrylase from Bacillus subtilis, Butyrate Kinase fromGeobacillus sp. and UbiD Protein from Saccharomyces cerevisiae

A pETDuet™-1 co-expression vector, carrying the UbiD gene fromSaccharomyces cerevisiae (Uniprot Accession Number Q03034) and the UbiXgene from Escherichia coli (Uniprot Accession Number P0AG03) (Example6), was used to produce and purify UbiD protein according to theprotocol described in Example 1. The phosphotransbutyrylase fromBacillus subtilis and the butyrate kinase from Geobacillus sp. werepurified as described in Example 4.

The enzymatic assays were conducted in a total reaction volume of 0.3ml.

The standard reaction mixture contained:

50 mM Tris-HCl pH 7.5

10 mM 3-methylcrotonyl-CoA

10 mM MgCl₂

10 mM NaCl

10 mM potassium phosphate buffer pH 7.5.

10 mM ADP

0.02 mg/ml purified phosphotransbutyrylase from B. subtilis

0.02 mg/ml purified butyrate kinase from Geobacillus sp.

1 mg/ml purified UbiD from S. cerevisiae

Catalysis was conducted at 30° C. during 18 h.

A series of control assays were performed in parallel in which either noUbiD protein (control A) or phosphotransbutyrylase (control B) orbutyrate kinase (control C) were added or no substrate was added(control D). After the incubation period, the isobutene produced in theheadspace was analysed by Gas Chromatography (GC) equipped FlameIonization Detector (FID). One ml of the headspace gas phase wasseparated and analysed according to the method described in Example 2.An overlay of typical chromatogram obtained for the whole enzymaticassay, and the corresponding controls is shown on FIG. 39.

The highest production of isobutene was observed in the assay comprisedphosphotransbutyrylase, butyrate kinase and UbiD protein. The controlassay without phosphotransbutyrylase (control B) and control assaywithout butyrate kinase (control C) also showed a significant productionof isobutene. These results could be explained by spontaneous hydrolysisof 3-methylcrotonyl-CoA into 3-methylcrotonic acid. Enzymatic productionof isobutene from 3-methylcrotonyl-CoA can thus be achieved by threeconsecutive steps, through the formation of 3-methylcrotonyl phosphateand 3-methylcrotonic acid as intermediates.

Example 8 In Vitro Screening of the UbiD Proteins for theDecarboxylation of 3-Methylcrotonic Acid Into Isobutene

Several genes coding for UbiD protein were codon optimized for theexpression in E. coli and synthesized by GeneArt® (Thermofisher). Thecorresponding enzymes were purified according to the procedure describedin Example 1. The list of the studied enzymes is shown in Table G.

Enzymatic assays were carried out in 2 ml glass vials (Interchim) underthe following conditions:

50 mM Tris-HCl buffer pH 7.5

20 mM NaCl

10 mM MgCl2

1 mM DTT

50 mM 3-methylcrotonic acid

1 mg/ml purified UbiD protein

100 μl lysate contained UbiX protein from E. coli

Total volume of the assays were 300 μl.

A series of control assays were performed in parallel, in which eitherno UbiD protein was added, or no enzymes were added (Table G).

The vials were sealed and incubated for 60 min at 30° C. The assays werestopped by incubating for 2 min at 80° C. and the isobutene formed inthe reaction headspace was analysed by Gas Chromatography (GC) equippedwith Flame Ionization Detector (FID), according to the proceduredescribed in Example 2.

The results of the GC analysis are shown in Table G. No isobuteneproduction was observed in control reactions. These results show thatall the UbiD proteins, studied under the conditions of this screeningassay, were able to perform the decarboxylation of 3-methylcrotonic acidinto isobutene in presence of E. coli cell lysate contained UbiXprotein.

TABLE G Isobutene produced, Candidate UbiD protein Assay compositionarbitrary units Saccharomyces cerevisae UbiD protein alone 9 (UniprotAccession UbiD protein + Cell lysate 945 Number: Q03034) contained UbiXprotein Sphaerulina musiva (Uniprot UbiD protein alone 70 AccessionNumber: M3DF95) UbiD protein + Cell lysate 3430 contained UbiX proteinPenicillium roqueforti (Uniprot UbiD protein alone 34 Accession Number:W6QKP7) UbiD protein + Cell lysate 1890 contained UbiX protein Hypocreaatroviridis (Uniprot UbiD protein alone 60 Accession Number: G9NLP8)UbiD protein + Cell lysate 5200 contained UbiX protein Fusariumoxysporum sp. UbiD protein alone 13 lycopersici (Uniprot Accession UbiDprotein + Cell lysate 1390 Number: W9LTH3) contained UbiX proteinSaccharomyces kudriavzevii UbiD protein alone 10 (Uniprot AccessionNumber: UbiD protein + Cell lysate 920 J8TRN5) contained UbiX protein«No UbiD control»: Cell lysate contained UbiX protein alone 0 Controlwithout any enzymes 0

Example 9 Conversion of 3-Methylcrotonyl-CoA and Acetate into3-Methylcrotonic Acid and acetyl-CoA Catalysed by Coenzyme A Transferasefrom Megasphaera sp

The enzyme was produced and purified according to the proceduredescribed in Example 1.

The enzymatic assays were conducted in a total reaction volume of 0.2 ml

The standard reaction mixture contained:

50 mM Tris-HCl buffer pH 7.5

5 mM 3-methylcrotonyl-CoA

10 mM sodium acetate

10 mM MgCl₂

10 mM NaCl

3 mg/ml purified CoA-transferase from Megasphaera sp. (Uniprot AccessionNumber: S7HFR5).

Control assays were performed in which either no enzyme was added, or no3-methylcrotonyl-CoA was added. The assays were incubated for 6 h at 30°C. The assays were stopped by adding 100 μl MeCN in the medium. Thesamples were centrifuged, filtered through a 0.22 μm filter and theclarified supernatants were transferred into a clean vial for theHPLC-based analysis.

HPLC analysis was performed using 1260 Inifinity LC System (Agilent),equipped with a column heating module and UV detector (260 nm). 5 μl ofsamples were separated on Zorbax SB-Aq column (250×4.6 mm, 5 μm particlesize, column temp. 30° C.), with a mobile phase flow rate of 1.5 ml/min.The separation was performed using mixed A (H₂O containing 8.4 mMsulfuric acid) and B (acetonitrile) solutions in a linear gradient (0% Bat initial time 0 min→70% B at 8 min). In these conditions, theretention time of 3-methylcrotonyl-CoA, 3-methylcrotonic acid andacetyl-CoA were 5.22 min, 5.70 min and 4.25 min, respectively.

Significant amounts of acetyl-CoA and 3-methylcrotonic acid wereobserved in the enzyme assay while none of the two compounds was notobserved in control Significant amounts of acetyl-CoA and3-methylcrotonic acid were observed in the enzyme assay while none ofthese two compounds was formed in control assays.

Typical chromatograms for enzymatic and control assays are shown on FIG.40.

Example 10 Enzymatic Decarboxylation of 3-Methylcrotonic Acid IntoIsobutene Catalyzed in the Presence of a Lysate Containing UbiX Proteinand with Purified Decarboxylase

0.5 M stock solution of 3-methylcrotonic acid was prepared in water andadjusted to pH 7.0 with 10 M solution of NaOH.

Proteins encoded by the aroY gene and one protein annotated as UbiDprotein were produced according to the procedure described in Example 1.

Enzymatic assays were carried out in 2 ml glass vials (Interchim) underthe following conditions:

50 mM potassium phosphate buffer pH 7.5

20 mM NaCl

10 mM MgCl₂

5 mM DTT

50 mM 3-methylcrotonic acid

1 mg/ml purified AroY or UbiD protein

50 μl lysate contained UbiX protein

Total volume of the assays were 300 μl.

A series of control assays were performed in parallel (Table H).

The vials were sealed and incubated for 120 min at 30° C. The assayswere stopped by incubating for 2 min at 80° C. and the isobutene formedin the reaction headspace was analysed by Gas Chromatography (GC)equipped with Flame Ionization Detector (FID).

For the GC analysis, one ml of the headspace gas was separated in aBruker GC-450 system equipped with a GS-alumina column (30 m×0.53 mm)(Agilent) using isothermal mode at 130° C. Nitrogen was used as carriergas with a flow rate of 6 ml/min.

The enzymatic reaction product was identified by comparison with anisobutene standard. Under these GC conditions, the retention time ofisobutene was 2.42 min.

A significant production of isobutene from 3-methylcrotonic acid wasobserved in the combined assays (AroY or UbiD protein+UbiX protein).Incubation of lysate containing UbiX protein alone did not result inisobutene production. These data indicate that the proteins encoded byaroY gene in association with UbiX protein can catalyze thedecarboxylation of 3-methylcrotonic acid into isobutene.

TABLE H Isobutene production, Assay composition arbitrary units AroYprotein from K. pneumoniae 10.5 (Uniprot Acession Number: B9A9M6) +lysate contained UbiX protein from E. coli + substrate AroY protein fromK. pneumoniae 0 (Uniprot Acession Number: B9A9M6) + substrate UbiDprotein from E. cloacae (Uniprot 8 Acession Number: V3DX94) + lysate,contained UbiX protein from E. coli + substrate UbiD protein from E.cloacae (Uniprot 0 Acession Number: V3DX94) + substrate AroY proteinfrom Leptolyngbya sp. 5.5 (Uniprot Acession Number: A0A0S3U6D8) +lysate,contained UbiX protein from E. coli + substrate AroY protein fromLeptolyngbya sp. 0 (Uniprot Acession Number: A0A0S3U6D8) + substrateAroY protein from Phascolarctobacterium 5.5 sp. (Uniprot AcessionNumber: R6I1V6) + lysate, contained UbiX protein from E. coli +substrate AroY protein from Phascolarctobacterium 0 sp. (UniprotAcession Number: R6I1V6) + substrate Lysate contained UbiX protein fromE. 0 coli + substrate

Example 11 Enzyme-Catalyzed Dehydration of3-Hydroxy-3-Methylglutaryl-CoA into 3-Methylglutaconyl-CoA

The genes coding for 3-hydroxyacyl-CoA dehydratases (also termedenoyl-CoA hydratases, abbreviated in the following by ECH) (Table I)were synthesized and the corresponding enzymes were further producedaccording to the procedure described in Example 1. Stock solution of 20mM 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) was prepared in water. Theenzymatic assays were conducted in total volume of 0.2 ml in thefollowing conditions:

50 mM Tris-HCl buffer pH 7.5

100 mM NaCl

2 mM of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA)

0.1 mg/ml purified 3-hydroxyacyl-CoA dehydratase.

Enzymatic assays were started by adding the 20 μl of 20 mM substrate,were run for 10 min at 30° C. run for and stopped by adding 100 μL ofacetonitrile in the reaction medium. All the enzymatic assays were

performed in duplicate. The samples were then centrifuged, filteredthrough a 0.22 μm filter and the clarified supernatants were transferredinto a clean vial for HPLC based analysis.

The analysis was performed using 1260 Inifinity LC System (Agilent),equipped with column heating module and UV detector (260 nm). 5 μl ofsamples were separated on Zorbax SB-Aq column (250×4.6 mm, 5 μm particlesize, column temp. 30° C.), with a mobile phase flow rate of 1.5 ml/min.The separation was performed using mixed A (H₂O containing 8.4 mMsulfuric acid) and B (acetonitrile) solutions in a linear gradient (0% Bat initial time 0 min→70% B at 8 min). In these conditions, theretention time of HMG-CoA, 3-methylglutaconyl-CoA (MG-CoA) and freecoenzyme A were respectively 4.26 min, 4.76 min and 3.96 min. FIG. 41shows 3-methylglutaconyl-CoA (MG-CoA) peak areas obtained from theHPLC-based analysis.

TABLE I Enzyme's abbreviation Source and Uniprot Accession Numbers LiuC3-hydroxybutyryl-CoA dehydratase from Myxococcus xanthus (Q1D5Y4) ECH UmPutative enoyl-CoA hydratase from Ustilago maydis (Q4PEN0) ECH BsMethylglutaconyl-CoA hydratase from Bacillus sp. GeD10 (N1LWG2) ECH LIMethylglutaconyl-CoA hydratase from Labilithrix luteola (A0A0K1PN19) ECHPa Putative isohexenylglutaconyl-CoA hydratase from Pseudomonasaeruginosa (Q9HZV7) ECH Ms Enoyl-CoA hydratase from Marinobactersantoriniensis (M7CV63) ECH Ab Enoyl-CoA hydratase from Acinetobacterbaumannii (A0A0D5YDD4) ECH Pp Isohexenylglutaconyl-CoA hydratase fromPseudomonas pseudoalcaligenes (L8MQT6)

Example 12 Microorganism for the Production of Isobutene from acetyl-CoAvia 3-Methylcrotonic Acid

This example shows the direct production of isobutene by a recombinantE. coli strain which expresses exogenous genes, thereby constituting theisobutene pathway. Like most organisms, E. coli converts glucose toacetyl-CoA. The enzymes used in this study to convert acetyl-CoA intoisobutene via 3-methylcrotonic acid (FIG. 42) are summarized in Table J.

TABLE J Uniprot Gene Accession Step Enzyme abbreviation NCB referencenumber XIII Acetyl-CoA thIA WP_ P45359 transferase from 010966157.1Clostridium acetobulyticum (ThIA) IX Hydroxymethylglutaryl- mvaS WP_Q9FD71 CoA synthase from 002357756.1 Enterococcus faecalis (MvaS) VIIIIsohexenylglutaconyl- ppKF707_ WP_ L8MQT6 CoA hydratase from 3831004422368.1 Pseudomonas pseudoalcaligenes KF707 (ECH) VII GlutaconateCoA- MXAN_ WP_ Q1D4I3 transferase from 4264 011554268.1 Myxococcusxanthus MXAN_ WP_ Q1D4I4 (AibA/B) 4265 011554267.1 VI Acyl-CoA tesB WP_P0AGG2 thioesterase 2 from 000075876.1 Escherichia coli (TesB) I Ferulicacid FDC1 XP_ G9NLP8 decarboxylase 013946967.1 from Hypocrea atroviridis(UbiD) Flavin prenyl ubiX WP_ P0AG03 transferase from 000825700.1Escherichia coli (UbiX)

Expression of Isobutene Biosynthetic Pathway in E. coli

All the corresponding genes were codon optimized for the expression inE. coli and synthesized by GeneArt® (Life Technologies), except the geneencoding for UbiX protein which was directly amplified from the genomicDNA of E. coli MG1655. The modified version of pUC18 (New EnglandBiolabs), containing a modified Multiple Cloning Site (pUC18 MCS) (WO2013/007786), was used for the overexpression of the ubiX gene. Thisplasmid conferred ampicillin resistance to the recombinant strain. Theconstructed vector was named pGB 5796 and the corresponding nucleotidicsequence is indicated in Table K.

TABLE K Plasmid name Nucleotidic sequence pGB 5796tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgccAAGCTTGCGGCCGCGGGGTTAATTAATTTCTCCTCTTTAATAAAGCAAATAAATTTTTTATGATTTGTTTAAACCTAGGCATGCCtctagaTTAttaTGCGCCCTGCCAGCGGGCAAAGAGATCTTCAGGAAGGGTTATCGCAAACTGGTCAAGAACACGATTAACCGTCTGATTTATCACATCATCAAGGGATTGCGGGCGATGATAAAACGCCGGAACGGGAGGCATAATCACCGCACCGATTTCTGCCGCCTGAGTCATTAAACGCAGATGGCCTAAGTGCAATGGTGTTTCACGCACGCAGAGCACCAACGGGCGACGCTCTTTCAGCACCACATCTGCCGCACGGGTCAGTAAGCCATCAGTATAGCTATGGACAATGCCGGAAAGGGTTTTGATTGAACAGGGTAAAATCACCATCCCCAGCGTCTGGAAAGAACCGGAAGAGATGCTGGCGGCAATATCGCGCGCATCGTGCGTGACATCGGCTAATGCCTGCACTTCGCGCAGAGAAAAATCCGTTTCGAGGGATAAGGTCTGGCGCGCTGCCTGGCTCATCACCAGATGCGTTTCGATATCTGTGACATCGCGCAGAACCTGTAATAAGCGCACGCCATAAATCGCGCCGCTGGCACCGCTGATGCCTACAATGAGTCGTTTcatAAAAAAAATGTATATCTCCTTCggtaccGAGCTCGAACCTGCAGGAATTCgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacggg ataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc (SEQ ID NO: 93)

An expression vector containing the origin of replication pSC was usedfor the expression of the genes: thIA, MvaS, ppKF707_3831,MXAN_4264/MXAN_4265, FDC1. This plasmid conferred spectinomycinresistance to the recombinant strain. The constructing vector was namedpGB 5771 and the corresponding nucleotidic sequence is indicated inTable L.

TABLE L Plasmid name Nucleotidic sequence pGB 5771ctcactactttagtcagttccgcagtattacaaaaggatgtcgcaaacgctgtttgctcctctacaaaacagaccttaaaaccctaaaggcttaagtagcaccctcgcaagctcgggcaaatcgctgaatattccttttgtctccgaccatcaggcacctgagtcgctgtctttttcgtgacattcagttcgctgcgctcacggctctggcagtgaatgggggtaaatggcactacaggcgccttttatggattcatgcaaggaaactacccataatacaagaaaagcccgtcacgcttctcagggcgttttatggcgggtctgctatgtggtgctatctgactttttgctgttcagcagttcctgccctctgattttccagtctgaccctagtcaaggccttaagtgagtcgtattacggactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgCCCGGGGAACTATAgtttaaacTTTTCAATGAATTCATTTaaGCGGCCGCatcaatTCTAGAatttaaatagtcaaaagcctccgaccggaggcttttgactgACCTATTGACAATTAAAGGCTAAAATGCTATAATTCCACtaatagaaataattttgtttaactttaggtctctatcgtaaGAAGGAGATATatgaaagaagtggtgattgccagcgcagttcgtaccgcaattggtagctatggtaaaagcctgaaagatgttccggcagttgatctgggtgcaaccgcaattaaagaagcagttaaaaaagccggtattaaaccggaagatgtgaacgaagttattctgggtaatgttctgcaagcaggtctgggtcagaatccggcacgtcaggcctcgtttaaagcaggtctgccggttgaaattccggcaatgaccattaacaaagtttgtggtagcggtctgcgtaccgttagcctggcagcacagattatcaaagccggtgatgcagatgttattattgccggtggtatggaaaatatgagccgtgcaccgtatctggcaaataatgcacgttggggttatcgtatgggtaatgccaaatttgtggatgagatgattaccgatggtctgtgggatgcctttaatgattatcacatgggtattaccgcagagaatattgcagaacgttggaatattagccgtgaagaacaggatgaatttgcactggcaagccagaaaaaagcagaagaagcaattaaaagcggtcagttcaaagatgaaattgtgccggttgttatcaaaggtcgtaaaggtgaaaccgttgttgataccgatgaacatccgcgttttggtagcaccattgaaggtctggcaaaactgaaaccggcattcaaaaaagatggcaccgttaccgcaggtaatgcaagcggtctgaatgattgtgcagcagttctggttattatgagcgcagaaaaagcaaaagaactgggtgttaaaccgctggcaaaaattgtgagctatggtagtgccggtgttgatccggcaattatgggttatggtccgttttatgcaaccaaagcagcaattgaaaaagcaggttggaccgttgatgaactggatctgattgaaagcaatgaagcatttgcagcacagagcctggcagttgcaaaagacctgaaattcgatatgaataaagtgaatgtgaatggcggtgcaattgccctgggtcatccgattggtgcaagcggtgcacgtattctggttaccctggttcatgcaatgcagaaacgtgatgcaaaaaaaggtctggccaccctgtgtattggtggtggtcagggcaccgcaattctgctggaaaaatgctaataagcttGAAGGAGATATAATGACCATTGGTATTGATAAAATCAGCTTTTTCGTGCCTCCGTACTATATTGATATGACCGCACTGGCCGAAGCACGTAATGTTGATCCGGGTAAATTTCATATTGGTATTGGTCAGGATCAGATGGCCGTTAATCCGATTAGCCAGGATATTGTTACCTTTGCAGCAAATGCAGCAGAAGCAATTCTGACCAAAGAAGATAAAGAGGCCATTGATATGGTTATTGTTGGCACCGAAAGCAGCATTGATGAAAGCAAAGCAGCAGCAGTTGTTCTGCATCGTCTGATGGGTATTCAGCCGTTTGCACGTAGCTTTGAAATTAAAGAAGCATGTTACGGAGCAACCGCAGGTCTGCAACTGGCAAAAAATCATGTTGCACTGCATCCGGATAAAAAAGTTCTGGTTGTTGCAGCAGATATTGCCAAATATGGTCTGAATAGCGGTGGTGAACCGACCCAGGGTGCCGGTGCAGTTGCAATGCTGGTTGCAAGCGAACCGCGTATTCTGGCACTGAAAGAAGATAATGTTATGCTGACCCAGGATATTTATGATTTTTGGCGTCCGACCGGTCATCCGTATCCGATGGTTGATGGTCCGCTGAGCAATGAAACCTATATTCAGAGCTTTGCACAGGTGTGGGATGAACATAAAAAACGTACCGGTCTGGATTTCGCAGATTATGATGCACTGGCATTTCATATCCCGTATACCAAAATGGGTAAAAAAGCACTGCTGGCCAAAATTAGCGATCAGACCGAAGCCGAACAAGAACGCATTCTGGCACGTTATGAAGAAAGCATTGTTTATAGCCGTCGTGTGGGTAATCTGTATACCGGTAGCCTGTATCTGGGTCTGATTAGCCTGCTGGAAAATGCAACCACCCTGACCGCAGGTAATCAGATTGGTCTGTTTAGCTATGGTAGCGGTGCCGTTGCAGAATTTTTCACAGGTGAACTGGTTGCAGGTTATCAGAATCATCTGCAAAAAGAAACCCATCTGGCACTGCTGGATAATCGTACCGAACTGAGCATTGCAGAATATGAAGCAATGTTTGCAGAAACCCTGGATACCGATATTGATCAGACCCTGGAAGATGAACTGAAATATAGCATTAGCGCCATTAATAACACCGTGCGTAGCTATCGTAACTAATAAggtaGAAGGAGATATACATatgagtcaggcgctaaaaaatttactgacattgttaaatctggaaaaaattgaggaaggactctttcgcggccagagtgaag atttaggtttacgccaggtgtttggcggccaggtcgtgggtcaggccttgtatgctgcaaaagagacGgtccctgaagaAcggctggtacattcgtttcacagctactttcttcgccctggcgatagtaagaagccgattatttatgatgtcgaaacgctgcgtgacggtaacagcttcagcgcccgccgggttgctgctattcaaaacggcaaaccgattttttatatgactgcctctttccaggcaccagaagcgggtttcgaacatcaaaaaacaatgccgtccgcgccagcgcctgatggcctcccttcggaaacgcaaatcgcccaatcgctggcgcacctgctgccgccagtgctgaaagataaattcatctgcgatcgtccgctggaagtccgtccggtggagtttcataacccactgaaaggtcacgtcgcagaaccacatcgtcaggtgtggatTcgcgcaaatggtagcgtgccggatgacctgcgcgttcatcagtatctgctcggttacgcttctgatcttaacttcctgccggtagctctacagccgcacggcatcggttttctcgaaccggggattcagattgccaccattgaccattccatgtggttccatcgcccgtttaatttgaatgaatggctgctgtatagcgtggagagcacctcggcgtccagcgcacgtggctttgtgcgcggtgagttttatacccaagacggcgtactggttgcctcgaccgttcaggaaggggtgatgcgtaatcacaattaataag aacGAAGGAGATATAAtgAAAACCGCACGTTGGTGTAGCCTGGAAGAAGCAGTTGCAAGCATTCCGGATGGTGCAAGCCTGGCAACCGGTGGTTTTATGCTGGGTCGTGCACCGATGGCACTGGTTATGGAACTGATTGCACAGGGTAAACGTGATCTGGGTCTGATTAGCCTGCCGAATCCGCTGCCAGCAGAATTTCTGGTTGCCGGTGGTTGTCTGGCTCGTCTGGAAATTGCATTTGGTGCACTGAGTCTGCAAGGTCGTGTTCGTCCGATGCCGTGTCTGAAACGTGCAATGGAACAGGGCACCCTGGCATGGCGTGAACATGATGGTTATCGTGTTGTTCAGCGTCTGCGTGCAGCAAGCATGGGTCTGCCGTTTATTCCGGCACCGGATGCAGATGTTAGCGGTCTGGCACGTACCGAACCGCCTCCGACCGTTGAAGATCCGTTTACCGGTCTGCGTGTTGCAGTTGAACCGGCATTTTATCCGGATGTTGCACTGCTGCACGCACGTGCAGCCGATGAACGTGGTAATCTGTATATGGAAGATCCGACCACCGATCTGCTGGTTGCGGGTGCAGCAAAACGTGTTATTGCAACCGTTGAAGAACGTGTTGCAAAACTGCCTCGTGCAACCCTGCCTGGTTTTCAGGTTGATCGTATTGTTCTGGCACCGGGTGGTGCACTGCCGACCGGTTGTGCAGGTCTGTATCCGCATGATGATGAAATGCTGGCACGTTATCTGAGCCTGGCAGAAACCGGTCGTGAAGCCGAATTTCTGGAAACCCTGCTGACCCGTCGTGCAGCATAATGAggatccGAAGGAGATATACATAtgAGCGCAACCCTGGATATTACACCGGCAGAAACCGTTGTTAGCCTGCTGGCACGTCAGATTGATGATGGTGGTGTTGTTGCAACCGGTGTTGCAAGTCCGCTGGCAATTCTGGCCATTGCAGTTGCACGTGCCACCCATGCACCGGATCTGACCTATCTGGCATGTGTTGGTAGCCTGGACCCGGAAATTCCGACCCTGCTGCCGAGCAGCGAAGACCTGGGTTATCTGGATGGTCGTAGCGCAGAAATTACCATTCCGGACCTGTTTGATCATGCACGTCGTGGTCGTGTTGATACCGTTTTTTTTGGTGCAGCCGAAGTTGATGCCGAAGGTCGTACCAATATGACCGCAAGCGGTAGTCTGGATAAACCGCGTACCAAATTTCCGGGTGTTGCCGGTGCAGCCACCCTGCGTCAGTGGGTTCGTCGTCCGGTTCTGCTGGTTCCGCGTCAGAGCCGTCGTAATCTGGTTCCGGAAGTTCAGGTTGCAACCACCCGTGATCCGCGTCGTCCGGTGACCCTGATTAGCGATCTGGGTGTTTTTGAACTGGGTGCAAGCGGTGCACGTCTGCTGGCACGCCATCCGTGGGCAAGCGAAGAACATATTGCAGAACGTACCGGTTTTGCATTTCAGGTTAGCGAAGCACTGAGCGTTACCAGCCTGCCGGATGCACGTACCGTTGCAGCAATTCGTGCAATTGATCCGCATGGCTATCGTGATGCACTGGTTGGTGCATAATTAgtcagaaggagatataCATATGAGCCTGCCGCATTGTGAAACCCTGCTGCTGGAACCGATTGAAGGTGTTCTGCGTATTACCCTGAATCGTCCGCAGAGCCGTAATGCAATGAGCCTGGCAATGGTTGGTGAACTGCGTGCAGTTCTGGCAGCAGTTCGTGATGATCGTAGCGTTCGTGCACTGGTTCTGCGTGGTGCAGATGGTCATTTTTGTGCCGGTGGTGATATTAAAGATATGGCAGGCGCACGTGCAGCCGGTGCAGAAGCATATCGTACACTGAATCGTGCATTTGGTAGCCTGCTGGAAGAAGCACAGGCAGCACCGCAGCTGCTGGTTGCACTGGTTGAAGGTGCCGTTCTGGGTGGTGGTTTTGGTCTGGCATGTGTTAGTGATGTTGCAATTGCAGCAGCAGATGCACAGTTTGGTCTGCCGGAAACCAGCCTGGGTATTCTGCCTGCACAGATTGCACCGTTTGTTGTTCGTCGTATTGGTCTGACCCAGGCACGTCGTCTGGCACTGACCGCAGCACGTTTTGATGGTCGTGAAGCACTGCGTCTGGGTCTGGTTCATTTTTGTGAAGCAGATGCAGATGCACTGGAACAGCGTCTGGAAGAAACCCTGGAACAGCTGCGTCGTTGTGCACCGAATGCAAATGCAGCAACCAAAGCACTGCTGCTGGCAAGCGAAAGCGGTGAACTGGGTGCACTGCTGGATGATGCAGCACGTCAGTTTGCCGAAGCAGTTGGTGGTGCAGAAGGTAGCGAAGGCACCCTGGCATTTGTTCAGAAACGTAAACCGGTTTGGGCACAGTAATAAtgaaagagaccagcctgatacagattaaatcagaacgcagaagcggtctgataaaacagaatttgcctggcggcagtagcgcggtggtcccacctgaccccatgccgaactcagaagtgaaacgccgtagcgccgatggtagtgtggggtcaccccatgcgagagtagggaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaactACTAGAatttaaatagtcaaaagcctccgaccggaggcttttgactgACCTATTGACAATTAAAGGCTAAAATGCTATAATTCCACtaatagaaataattttgtttaactttaggtctctatcgaccataaTTAATTAActttaagaaggagatataCATatgAGCAGCACCACCTATAAAAGCGAAGCATTTGATCCGGAACCGCCTCATCTGAGCTTTCGTAGCTTTGTTGAAGCACTGCGTCAGGATAATGATCTGGTGGATATTAATGAACCGGTTGATCCGGATCTGGAAGCAGCAGCAATTACCCGTCTGGTTTGTGAAACCGATGATAAAGCACCGCTGTTTAATAACGTGATTGGTGCAAAAGATGGTCTGTGGCGTATTCTGGGTGCACCGGCAAGCCTGCGTAGCAGCCCGAAAGAACGTTTTGGTCGTCTGGCACGTCATCTGGCACTGCCTCCGACCGCAAGCGCAAAAGATATTCTGGATAAAATGCTGAGCGCCAATAGCATTCCGCCTATTGAACCGGTTATTGTTCCGACCGGTCCGGTTAAAGAAAATAGCATTGAAGGCGAAAACATTGATCTGGAAGCCCTGCCTGCACCGATGGTTCATCAGAGTGATGGTGGCAAGTATATCCAGACCTATGGTATGCATGTTATCCAGAGTCCGGATGGTTGTTGGACCAATTGGAGCATTGCCCGTGCAATGGTTAGCGGTAAACGTACCCTGGCAGGTCTGGTTATTAGTCCGCAGCATATTCGTAAAATTCAGGATCAGTGGCGTGCAATTGGTCAAGAAGAAATTCCTTGGGCACTGGCATTTGGTGTTCCGCCTACCGCAATTATGGCAAGCAGTATGCCGATTCCGGATGGTGTTAGCGAAGCAGGTTATGTTGGTGCAATTGCCGGTGAACCGATTAAACTGGTTAAATGCGATACCAACAATCTGTATGTTCCGGCAAATAGCGAAATTGTTCTGGAAGGCACCCTGAGCACCACCAAAATGGCACCGGAAGGTCCGTTTGGTGAAATGCATGGTTATGTTTATCCGGGTGAAAGCCATCCGGGTCCGGTTTATACCGTTAACAAAATTACCTATCGCAACAATGCAATTCTGCCGATGAGCGCATGTGGTCGTCTGACCGATGAAACCCAGACCATGATTGGCACCCTGGCAGCAGCAGAAATTCGTCAGCTGTGTCAGGATGCAGGTCTGCCGATTACCGATGCATTTGCACCGTTTGTTGGTCAGGCAACCTGGGTTGCACTGAAAGTTGATACCAAACGTCTGCGTGCAATGAAAACCAATGGTAAAGCATTTGCAAAACGTGTTGGTGATGTTGTGTTTACCCAGAAACCGGGTTTTACCATTCATCGTCTGATTCTGGTTGGTGATGATATTGATGTGTATGACGATAAAGATGTGATGTGGGCATTTACCACCCGTTGTCGTCCGGGTACAGATGAAGTTTTTTTTGATGATGTTGTGGGCTTTCAGCTGATCCCGTATATGAGTCATGGTAATGCCGAAGCAATTAAAGGTGGTAAAGTTGTTAGTGATGCACTGCTGACCGCAGAATATACCACCGGTAAAGATTGGGAAAGCGCAGATTTCAAAAACAGCTATCCGAAAAGCATCCAGGATAAAGTTCTGAATAGCTGGGAACGCCTGGGTTTCAAAAAACTGGATTAATAACCATGGttataagagagaccagcctGACTCCTGTTGATAGATCCAGTAATGACCTCAGAACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCCGCCGGGCGTTTTTTATTGGTGAGAATaactACTAGTtggcggGCGGCCGCttagctCTGCAGatgagaaattcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttAAGCTTcttagaataGCTCTTCTATGaggtggcacttttcggggaaaGATATCcgcatatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatcGGTACCtttcatgatatatctcccaatttgtgtagggcttattatgcacgcttaaaaataataaaagcagacttgacctgatagtttggctgtgagcaattatgtgcttagtgcatctaacgcttgagttaagccgcgccgcgaagcggcgtcggcttgaacgaattgttagacattatttgccgactaccttggtgatctcgcctttcacgtagtggacaaattcttccaactgatctgcgcgcgaggccaagcgatcttcttcttgtccaagataagcctgtctagcttcaagtatgacgggctgatactgggccggcaggcgctccattgcccagtcggcagcgacatccttcggcgcgattttgccggttactgcgctgtaccaaatgcgggacaacgtaagcactacatttcgctcatcgccagcccagtcgggcggcg agttccatagcgttaaggtttcatttagcgcctcaaatagatcctgttcaggaaccggatcaaagagttcctccgccgctggacctaccaaggcaacgctatgttctcttgcttttgtcagcaagatagccagatcaatgtcgatcgtggctggctcgaagatacctgcaagaatgtcattgcgctgccattctccaaattgcagttcgcgcttagctgg ataacgccacgg aatgatgtcgtcgtgcacaacaatggtgacttctacagcgcggagaatctcgctctctccaggggaagccgaagtttccaaaaggtcgttgatcaaagctcgccgcgttgtttcatcaagccttacggtcaccgtaaccagcaaatcaatatcactgtgtggcttcaggccgccatccactgcggagccgtacaaatgtacggccagcaacgtcggttcgagatggcgctcgatgacgccaactacctctgatagttgagtcgatacttcggcgatcaccgcttccctcatgatgtttaactttgttttagggcgactgccctgctgcgtaacatcgttgctgctccataacatcaaacatcgacccacggcgtaacgcgcttgctgcttggatgcccgaggcatagactgtaccccaaaaaaacagtcataacaagccatgaaaaccgccacGAGCTCctgtcagaccaagtttacgagctcgcttggactcctgttgatagatccagtaatgacctcagaactccatctggatttgttcagaacgctcggttgccgccgggcgttttttattggtgagaatccaagcactagggacagtaagacgggtaagcctgttgatgataccgctgccttactgggtgcattagccagtctgaatgacctgtcacgggataatccgaagtggtcagactggaaaatcagagggcaggaactgctgaacagcaaaaagtcagatagcaccacatagcagacccgccataaaacgccctgagaagcccgtgacgggcttttcttgtattatgggtagtttccttgcatgaatccataaaaggcgcctgtagtgccatttacccccattcactgccagagccgtgagcgcagcgaactgaatgtcacgaaaaagacagcgactcaggtgcctgatggtcggagacaaaaggaatattcagcgatttgcccgagcttgcgagggtgctacttaagcctttagggttttaaggtctgttttgtag aggagcaaacagcgtttgcgacatccttttgtaatactgcgg aactgactaaagtagtgagttatacacagggctgggatctattctttttatctttttttattctttctttattctataaattataaccacttgaatataaacaaaaaaaacacacaaaggtctagcggaatttacagagggtctagcagaatttacaagttttccagcaaaggtctagcagaatttacagatacccacaactcaaaggaaaaggacatgtaattatcattgactagcccatctcaattggtatagtgattaaaatcacctagaccaattgagatgtatgtctgaattagttgttttcaaagcaaatgaactagcgattagtcgctatgacttaacggagcatgaaaccaagctaattttatgctgtgtggcactactcaaccccacgattg aaaaccctacaaggaaagaacggacggtatcgttcacttataaccaatacgctcagatgatgaacatcagtagggaaaatgcttatggtgtattagctaaagcaaccagagagctgatgacgagaactgtggaaatcaggaatcctttggttaaaggctttgagattttccagtggacaaactatgccaagttctcaagcg aaaaattagaattagtttttagtgaagagatattgccttatcttttccagttaaaaaaattcataaaatataatctggaacatgttaagtcttttgaaaacaaatactctatgaggatttatgagtggttattaaaagaactaacacaaaagaaaactcacaaggcaaatatagagattagccttgatgaatttaagttcatgttaatgcttgaaaataactaccatgagtttaaaaggcttaaccaatgggttttgaaaccaataagtaaagatttaaacacttacagcaatatgaaattggtggttgataagcgaggccgcccgactgatacgttgattttccaagttgaactagatagacaaatggatctcgtaaccgaacttgagaacaaccagataaaaatgaatggtgacaaaataccaacaaccattacatcagattcctacctacgtaacggactaagaaaaacactacacgatgctttaactgcaaaaattcagctcaccagttttgaggcaaaatttttgagtgacatgcaaagtaagcatg atctcaatggttcgttctcatggctcacgcaaaaacaacgaaccacactagagaacatactggctaaatacggaaggatctgaggttcttatggctcttgtatctatcagtgaagcatcaagactaacaaacaaaagtagaacaactgttcaccgttagatatcaaagggaaaactgtccataagcacagatgaaaacggtgtaaaaaagatagatacatcagagcttttacgagtttttggtgcatttaaagctgttcaccatgaacagatcgacaatgtaacGCATGCaccgagcgcagcgagtcagtgagcgaggaagcggaacagcgcctg (SEQ ID NO: 94)

These recombinant pGBE 5771 and pGBE5796 plasmids were verified bysequencing.

MG1655 E. coli strain was made electrocompetent and was transformed withpGBE5771 and pGBE5796 or with the corresponding empty vectors (pUC18 MCSand pGB2021) in order to create negative controls. The strains thusproduced are summarized in Table M.

TABLE M Strain number Vectors Strain 1 (metabolic pUC18_MCS + pGB 2021pathway-free control), containing the empty vectors. Strain 2,expressing only pGB 5796 + pGB 2021 UbiX protein Strain 3, expressingthe pUC18_MCS + PGB 5771 whole metabolic pathway, without overexpressionof UbiX protein on plasmid. Strain 4, expressing the pGB 5796 + pGB 5771whole metabolic pathway, comprising overexpression of UbiX protein onplasmid.

The transformed cells were then plated on LB plates, supplied withampicillin (100 μg/ml) and spectinomycin (100 μg/ml). Plates wereincubated overnight at 30° C. Isolated colonies were used to inoculate1.4 ml of ZYM-5052 auto-inducing media (Studier FW, Prot. Exp. Pur. 41,(2005), 207-234) supplemented with ampicillin, spectinomycin and 0.5 mMflavin mononucleotide. These cultures were grown for 16 h at 30° C. and700 rpm shaking in 96 deep-well microplates. Then the cultures werecentrifuged and the pellets were resuspended in 0.4 ml of MS medium(Richaud C., Mengin-Leucreulx D., Pochet S., Johnson E J., Cohen G N.and Marlière P, The Journal of Biological Chemistry, 268, (1993),26827-26835) containing glucose (45 g/L), and MgSO₄ (1 mM). The cultureswere further incubated in 96 deep-well sealed microplates at 30° C., 700rpm shaking for 24 hours. The production of isobutene was stopped byincubating the microplates for 5 min at 80° C. and the isobutene formedin the reaction headspace was analysed by Gas Chromatography (GC)equipped with Flame Ionization Detector (FID). 100 μL of headspace gasesfrom each enzymatic reaction are injected in a Brucker GC-450 systemequipped with a Flame Ionization Detector (FID). Compounds present insamples were separated by chromatography using a GS-alumina column (30m×0.53 mm) (Agilent) using isothermal mode at 130° C. Nitrogen was usedas carrier gas with a flow rate of 6 ml/min. Upon injection, peak areasof isobutene were calculated; Table N.

TABLE N IBN production, Strain number Vectors arbitrary units Strain 1(metabolic pUC18_MCS + pGB 2021 950 pathway-free control), containingthe empty vectors Strain 2, expressing only pGB 5796 + pGB 2021 710 UbiXproteine Strain 3, expressing the pUC18_MCS + PGB 5771 625 wholemetabolic pathway, without overexpression of UbiX protein on plasmidStrain 4, expressing the pGB 5796 + pGB 5771 15192 whole metabolicpathway, comprising overexpression of UbiX protein on plasmid

1-36. (canceled)
 37. A recombinant organism or microorganism capable ofproducing isobutene, wherein said microorganism expresses polypeptidescomprising: a) at least one of: i. a CoA transferase (EC 2.8.3.-) and athioester hydrolase (EC 3.1.2.-); or ii. (a) a phosphatebutyryltransferase (EC 2.3.1.19) or a phosphate acetyltransferase (EC2.3.1.8) and (b) a phosphotransferase with a carboxy group as acceptor(EC 2.7.2.-) and b) a 3-methylcrotonic acid decarboxylase selected from:i. an FMN-dependent decarboxylase associated with an FMN prenyltransferase (EC 2.5.1.-); ii. an aconitate decarboxylase (EC 4.1.1.6);iii. a methylcrotonyl-CoA carboxylase (EC 6.4.1.4); iv. a geranoyl-CoAcarboxylase (EC 6.4.1.5); or v. a protocatechuate (PCA) decarboxylase(EC 4.1.1.63).
 38. The recombinant organism or microorganism of claim37, wherein the CoA transferase (EC 2.8.3.-) is selected from apropionate:acetate-CoA transferase (EC 2.8.3.1), an acetateCoA-transferase (EC 2.8.3.8) or a succinyl-CoA:acetate CoA-transferase(EC 2.8.3.18).
 39. The recombinant organism or microorganism of claim37, wherein the thioester hydrolase (EC 3.1.2.-) is selected from anacetyl-CoA hydrolase (EC 3.1.2.1), an ADP-dependent short-chain-acyl-CoAhydrolase (EC 3.1.2.18) or an acyl-CoA hydrolase (EC 3.1.2.20).
 40. Therecombinant organism or microorganism of claim 37, wherein thephosphotransferase with a carboxy group as acceptor (EC 2.7.2.-) isselected from a propionate kinase (EC 2.7.2.15), an acetate kinase (EC2.7.2.1), a butyrate kinase (EC 2.7.2.7) or a branched-chain-fatty-acidkinase (EC 2.7.2.14).
 41. The recombinant organism or microorganism ofclaim 37, wherein the recombinant organism or microorganism furtherexpresses a polypeptide selected from a methylcrotonyl-CoA carboxylase(EC 6.4.1.4) or a geranoyl-CoA carboxylase (EC 6.4.1.5).
 42. Therecombinant organism or microorganism of claim 41, wherein therecombinant organism or microorganism further expresses a polypeptideselected from a 3-methylglutaconyl-coenzyme A hydratase (EC 4.2.1.18), a3-hydroxyacyl-CoA dehydratase (EC 4.2.1.-) or an enoyl-CoA hydratase (EC4.2.1.-).
 43. The recombinant organism or microorganism of claim 42,wherein the recombinant organism or microorganism further expresses apolypeptide selected from a 3-hydroxy-3-methylglutaryl-CoA synthase. 44.The recombinant organism or microorganism of claim 37, wherein the3-methylcrotonic acid decarboxylase is a FMN-dependent decarboxylaseassociated with an FMN prenyl transferase (EC 2.5.1.-).
 45. Therecombinant organism or microorganism of claim 38, wherein the3-methylcrotonic acid decarboxylase is a FMN-dependent decarboxylaseassociated with an FMN prenyl transferase (EC 2.5.1.-).
 46. Therecombinant organism or microorganism of claim 39, wherein the3-methylcrotonic acid decarboxylase is a FMN-dependent decarboxylaseassociated with an FMN prenyl transferase (EC 2.5.1.-).
 47. Therecombinant organism or microorganism of claim 46, wherein the thioesterhydrolase (EC 3.1.2.-) is selected from an acetyl-CoA hydrolase (EC3.1.2.1), an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18)or an acyl-CoA hydrolase (EC 3.1.2.20).
 48. The recombinant organism ormicroorganism of claim 40, wherein the 3-methylcrotonic aciddecarboxylase is a FMN-dependent decarboxylase associated with an FMNprenyl transferase (EC 2.5.1.-).
 49. The recombinant organism ormicroorganism of claim 48, wherein the thioester hydrolase (EC 3.1.2.-)is selected from an acetyl-CoA hydrolase (EC 3.1.2.1), an ADP-dependentshort-chain-acyl-CoA hydrolase (EC 3.1.2.18) or an acyl-CoA hydrolase(EC 3.1.2.20).
 50. The recombinant organism or microorganism of claim41, wherein the 3-methylcrotonic acid decarboxylase is a FMN-dependentdecarboxylase associated with an FMN prenyl transferase (EC 2.5.1.-).51. The recombinant organism or microorganism of claim 50, wherein thethioester hydrolase (EC 3.1.2.-) is selected from an acetyl-CoAhydrolase (EC 3.1.2.1), an ADP-dependent short-chain-acyl-CoA hydrolase(EC 3.1.2.18) or an acyl-CoA hydrolase (EC 3.1.2.20).
 52. Therecombinant organism or microorganism of claim 42, wherein the3-methylcrotonic acid decarboxylase is a FMN-dependent decarboxylaseassociated with an FMN prenyl transferase (EC 2.5.1.-).
 53. Therecombinant organism or microorganism of claim 52, wherein the thioesterhydrolase (EC 3.1.2.-) is selected from an acetyl-CoA hydrolase (EC3.1.2.1), an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18)or an acyl-CoA hydrolase (EC 3.1.2.20).
 54. The recombinant organism ormicroorganism of claim 43, wherein the 3-methylcrotonic aciddecarboxylase is a FMN-dependent decarboxylase associated with an FMNprenyl transferase (EC 2.5.1.-).
 55. The recombinant organism ormicroorganism of claim 54, wherein the thioester hydrolase (EC 3.1.2.-)is selected from an acetyl-CoA hydrolase (EC 3.1.2.1), an ADP-dependentshort-chain-acyl-CoA hydrolase (EC 3.1.2.18) or an acyl-CoA hydrolase(EC 3.1.2.20).
 56. The recombinant organism or microorganism of claim44, wherein the 3-methylcrotonic acid decarboxylase is a FMN-dependentdecarboxylase associated with an FMN prenyl transferase (EC 2.5.1.-).57. The recombinant organism or microorganism of claim 56, wherein thethioester hydrolase (EC 3.1.2.-) is selected from an acetyl-CoAhydrolase (EC 3.1.2.1), an ADP-dependent short-chain-acyl-CoA hydrolase(EC 3.1.2.18) or an acyl-CoA hydrolase (EC 3.1.2.20).
 58. Therecombinant organism or microorganism of claim 45, wherein the3-methylcrotonic acid decarboxylase is a FMN-dependent decarboxylaseassociated with an FMN prenyl transferase (EC 2.5.1.-).
 59. Therecombinant organism or microorganism of claim 58, wherein the thioesterhydrolase (EC 3.1.2.-) is selected from an acetyl-CoA hydrolase (EC3.1.2.1), an ADP-dependent short-chain-acyl-CoA hydrolase (EC 3.1.2.18)or an acyl-CoA hydrolase (EC 3.1.2.20).